Methods and compositions for the selection and optimization of oligonucleotide tag sequences

ABSTRACT

Methods for selecting tag-oligonucleotide sequences for use in an in vitro nucleic acid assay. The selected tag sequences are useful for nucleic acid assay wherein interference between the nucleic acid sequences is the assay is to be controlled. Selected tag sequences are incorporated into nucleic acid assay to improve the performance of and/or minimize any interference between nucleic acids in the assay compared to untagged assays.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a reissue of U.S. Pat. No. 9,512,467, issued Dec. 6,2016, which is the U.S. national phase application of internationalApplication No. PCT/US2012/028797, filed on Mar. 12, 2012, which claimspriority from U.S. provisional application Ser. No. 61/451,285 filedMar. 10, 2011, the contents of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This present disclosure is directed to the field of nucleic acid-basedassays incorporating one or more tag sequences into nucleic acid(s) ofthe assay. More specifically, methods and compositions are describedrelated to the selection and optimization of oligonucleotide sequencesreferred to as “tags” for minimizing undesired nucleic acid interactionswithin the assay.

BACKGROUND

The use of short, user-selected (i.e., not defined by the target nucleicacid(s)) nucleic acid sequences (also known as “tags”) is a verypowerful technique for the design and development of novel nucleic acidassay formats. These nucleic acid formats include nucleic acidamplification, sequencing or other assay formats.

Nucleic acid amplification provides a means for making more copies of anucleic acid sequence that is relatively rare or unknown, foridentifying the source of nucleic acids, or for making sufficientnucleic acid to provide a readily detectable amount. Amplification isuseful in many applications, for example, in research, diagnostics, drugdevelopment, forensic investigations, environmental analysis, and foodtesting.

Typically, nucleic acid amplification uses one or more nucleic acidpolymerase and/or transcriptase enzymes to produce at least one copy,and preferably multiple copies of a target nucleic acid sequence and,optionally, a tag sequence.

Many methods for amplifying nucleic acid sequences in vitro are known,including polymerase chain reaction (PCR), ligase chain reaction (LCR),replicase-mediated amplification, strand-displacement amplification(SDA), “rolling circle” types of amplification, and variousTranscription Mediated Amplification (TMA) and reverse TMA (rTMA)methods. These known methods use different techniques to make amplifiedsequences, which usually are detected by using a variety of methods.See, for example, Schweitzer and Kingsmore, combining nucleic acidamplification and detection, current opinion in Biotechnology, 2001, 12,21-27. These methods can be exemplified by the following publications(each of which is hereby expressly incorporated by reference): PCR—U.S.Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; LCR—U.S. Pat. No.5,516,663 and EP 0320308 B1; Replicase-mediated amplification—U.S. Pat.No. 4,786,600; SDA—U.S. Pat. No. 5,422,252A and U.S. Pat. No. 5,547,861;Rolling circle types of amplification—U.S. Pat. No. 5,714,320 and U.S.Pat. No. 5,834,252; TMA—U.S. Pat. Nos. 4,868,105, 5,124,246, 5,130,238,5,399,491, 5,554,516 and 5,437,990, 5,824,518, US 2006-0046265 A1 and WO1988010315 A1; rTMA—US 2006-0046265.

Amplification methods may introduce nucleic acid sequences into thesequence being amplified. Some methods use modified primers to introducenon-target nucleic acid sequences to the sequence being amplified. Oneexample of a modified primer is a “tag” primer. A tag primer containstwo parts: (1) a “tag sequence” that is a nucleic acid sequence thatdoes not hybridize to the target nucleic acid sequence and (2) a primersequence that is a nucleic acid sequence that does hybridize to thetarget nucleic acid sequence. The tag sequence is located 5′ to theprimer sequence. The first round of amplification incorporates the tagsequence into the sequence being amplified. The second round ofamplification uses primers that are complementary to the tag sequence.

Anchored PCR is a modified PCR method that uses an “adaptor” primer toamplify a sequence which is only partially known. See, for example, Lohet al., 1989, Science 243(4888):217-20; Lin et al., 1990, Mol. Cell.Biol. 10(4): 1818-21).

Nested PCR may use primer(s) that contain a tag sequence unrelated tothe target nucleic acid's target sequence to amplify nucleic acid fromunknown target sequences in a reaction. See, for example, Sullivan etal, 1991, Electrophoresis 12(1):17-21; Sugimoto et al., 1991, Agric.Biol. Chem. 55(11):2687-92.

Other forms of amplification use a probe or probe set to introducenon-target priming sites located upstream and downstream of atarget-specific sequence and adapter sequence(s). See, for example, U.S.Pat. Nos. 6,812,005 and 6,890,741, Fan et al. The two probes that bindin close proximity on a target sequence may be ligated together beforebeing amplified by using the upstream and downstream universal primingsites.

Alternative assay methods may use probe hybridization and linear signalamplification by using a common sequence that is included in a varietyof target nucleic acid-specific probes (e.g., US 20070111200, Hudson etal.). This method uses a labeled cassette that contains a sequencecomplementary to the common sequence to detect multiple target nucleicacids.

One problem that has heretofore existed with the use of tags is the lackof a rigorous selection method to identify the best tag for a givenapplication. This has resulted in the use of less than optimal tags forparticular applications.

Another problem is that tags may engage in undesired cross-reactionswith other tags, primers, promoter providers, probes, amplicons, othertarget and non-target sequences and other such sequences in a givenassay.

Another problem is that tags may engage in undesired cross-reaction withsequences in test samples from known or unknown sources, such aspathogenic or non-pathogenic organisms, mammalian nucleic acids,contaminating nucleic acids from enzymes, side-products of nucleic acidamplification reactions, etc.

One of the unsolved problems with multiplexed detection or amplificationis the interference observed during multiplexing experiments, whichlimits the dynamic range, precision, and quantification characteristicsfor the target nucleic acids when present together in a sample.

In multiplex amplification reactions, a variety of undesired “sidereactions” can occur that ultimately degrade assay performance. Forinstance, in the Transcription-Mediated Amplification (TMA) context,primers and/or promoter based amplification oligomers directed towardsone target nucleic acid (or group of target nucleic acids) can interactwith primers and/or promoter based amplification oligomers directedtowards another target nucleic acid (or group of target nucleic acids),causing degraded performance of one, or the other or both amplificationsystems. This problem typically gets worse as the number ofamplification systems present in a multiplex reaction increases. Otherinteractions that reduce assay performance include amplificationoligomers interacting with one another or with other oligonucleotides inthe amplification reaction, such as probes, blockers and target captureoligomers (TCOs), and amplicons. This problem of negative interactionbetween nucleic acids in a system can be reduced or solved by eitherconverting all of the target specific primers/promoter providers in theassay or a portion of the target specific primers and/or promoterproviders in the assay into primers and/or promoter based amplificationoligomers containing tag sequences. Early rounds of amplification takeplace using these tagged amplification oligomers, thereby incorporatingthe tag sequences and their complements into the early amplificationproducts. Subsequent rounds of amplification can take place usingprimers and/or promoter-based amplification oligomers having targethybridizing sequences directed to the incorporated tag sequence. In thisway, the make-up of the subsequent round amplification oligomers iscontrolled by the user and undesired side reactions are reduced oreliminated. It is notable that the tag sequences used in two or moreseparate amplification oligomers can be the same sequence or differentsequences.

Another related problem is the lack of a convenient procedure toquantitatively or qualitatively measure the relative levels ofinterferences in a multiplexed reaction.

Additionally, competition for amplification reaction resources may occurin multiplex amplification reactions when the same tag sequence is usedfor multiple tagged amplification oligomers. For example, in multiplexamplification reactions, target nucleic acid present at high levels willconsume the amplification oligomers complementary to the tag sequencemuch faster than target nucleic acid present at low levels. Theinability to uniformly amplify the target nucleic acids due toamplification resource competition may lead to false negatives becausethe target nucleic acids present at low levels are not amplified to adetectable amount.

One possible solution to these problems is to create unique tagsequences for incorporation into one or more oligomers in an assay. Theunique tags are designed such that they do not interact with each other,or with any other sequences in the assay. In this way, an assayincorporating one or more tag sequences can proceed without reducedperformance caused by the undesired interaction of various nucleicacids, including tag sequences, present in the assay reaction.

Clearly, there are numerous problems in the art of using nucleic acidtag sequences in a nucleic acid assay. It would be desirable to have tagsequences and methods for identifying tag sequences that are useful in anucleic acid assay and that avoid the problems. It would be desirable tohave methods for rapidly identifying tag sequences for use in a nucleicacid assay.

SUMMARY OF THE INVENTION

It is, therefore, our object of the present invention to provide anidentification and selection method that can be used to generate uniquetag sequence sets. Thus, the invention encompasses methods to identifyand select nucleic acid tags for use in nucleic acid assays, sequencing,amplification, manipulation, interaction and other processing (sometimesreferred to herein genetically as “nucleic acid assays”). The inventionalso encompasses a method of minimizing interference between nucleicacid sequences present in an assay, including amplification assays,multiplex amplification assays, sequencing assays and the like. Inaddition, the invention also encompasses compositions which have beenselected by these methods.

The present invention encompasses a method for identifying a nucleicacid tag sequence for use in a nucleic acid assay, comprising: a)generating a pool of nucleic acid sequences, wherein the pool is atleast three nucleic acid sequences; b) screening the pool of nucleicacid sequences to identify two or more nucleic acid sequences have twoor more performance characteristics the; and c) selecting one or morenucleic acid sequences, each for use as tag sequence in a nucleic acidassay.

The invention further includes a method as described above, furthercomprising: d) comparing a nucleic acid sequences from the pool ofnucleic acid sequences against a database having one or more nucleicacid sequences to determine complementarity of the nucleic acidsequences from the pool of nucleic acid sequences to the database havingone or more sequences; e) generating a sub-pool of nucleic acidsequences, wherein the sub-pool is a collection of nucleic acidsequences with complementarity that is less than 95% to the nucleic acidsequence(s) in the database, that is less than 90% to the nucleic acidsequence(s) in the database; that is less than 80% to the nucleic acidsequence(s) in the database, that is less than 70% to the nucleic acidsequence(s) in the database, or that is less than 50% to the nucleicacid sequence(s) in the database; f) screening the sub-pool of nucleicacid sequences for one or more performance characteristics selected frommelting temperature, activity in an enzyme reaction, G-C content,hybridization energy, multimer formation, internal structure formation,G-quartet formation, and hairpin-stability; and g) selecting one or morenucleic acid sequences from the sub-pool for use as tag sequences in anucleic acid assay.

The invention further includes a method as described above, furthercomprising: h) synthesizing at least two different oligonucleotides foruse in a nucleic acid assay, wherein each of the synthesizedoligonucleotides has a tag sequence selected according to step g); andi) measuring for each of the different oligonucleotides synthesized instep h) one or more of the following performance characteristics: speedof reaction, limit of detection, interference, precision of replicates,performance against a specific target nucleic acid sequence, orperformance against multiple target nucleic acid sequences in a nucleicacid assay, and optionally comparing the measurements to themeasurements obtained for an untagged oligonucleotide; and j) selectingone or more of the nucleic acid tag sequences used in step i) for use ina nucleic acid assay.

The invention further includes a method as described above, furthercomprising the steps of: k) modifying the sequence of the tag sequenceincorporated into an oligonucleotide from step h) to obtain a modifiedtag sequence for incorporation into an oligonucleotide; l) measuring forthe oligonucleotide containing a modified tag sequence from step k) oneor more of the following performance characteristics: speed of reaction,limit of detection, interference, precision of replicates, performanceagainst a specific target nucleic acid sequence, or performance againstmultiple target nucleic acid sequences in a nucleic acid assay; and m)selecting one or more of the modified nucleic acid tag sequences used instep i) for use in a nucleic acid assay.

The invention further includes a method as described above, wherein themodification in step k) comprises systematically deleting nucleotidesfrom the tag sequence.

The invention further includes a method as described above, furthercomprising at step g), the steps of: (i) modifying the sequence of thetag sequence from step g); (ii) synthesizing an oligonucleotide tocontain the modified tag sequence; (iii) measuring for theoligonucleotide containing a modified tag sequence one or more of thefollowing performance characteristics: speed of reaction, limit ofdetection, interference, precision of replicates, performance against aspecific target nucleic acid sequence, or performance against multipletarget nucleic acid sequences in a nucleic acid assay; and (iv)selecting one or more of the modified nucleic acid tag sequences used instep (iii) for use in a nucleic acid assay.

The invention further includes a method as described above, wherein themodification in step g) sub-step (i) comprises systematically deletingnucleotides from the tag sequence.

The invention further includes a method as described above, wherein theperformance characteristic(s) is selected from the group consisting ofone or more amplification performance characteristic(s); interferencewith nucleic acids in the nucleic acid assay; interference with one ormore oligonucleotides in the nucleic acid assay; interference with oneor more target nucleic acids in the nucleic acid assay; interferencewith one or more amplicons in the nucleic acid assay; assayreproducibility; or quantification.

The invention further includes a method as described above, wherein theperformance characteristic is quantification.

The invention further includes a method as described above, wherein thequantification is real-time quantification.

The invention further includes a method as described above, wherein thequantification is end-point quantification.

The invention further includes a method as described above, wherein theperformance characteristic is a dynamic range for detecting targetnucleic acid; limit of detection; precision of replicates; or reactionkinetics.

The invention further includes a method as described above, wherein theperformance characteristics comprise reaction kinetics.

The invention further includes a method as described above, wherein anucleic acid sequence in the pool is used as a tag in a nucleic acidassay and reduces interference with a nucleic acid in the nucleic acidassay to about 95% or less compared to the amount of interferencepresent in an untagged assay.

The invention further includes a method as described above, wherein anucleic acid sequence in the pool is used as a tag in an in vitronucleic acid assay and accelerates reaction kinetics to about 105% ormore compared to the reaction kinetics in an untagged assay; slowsreaction kinetics to about 95% or less compared to the reaction kineticsin an untagged assay; increases sensitivity for a target nucleic acid sothat the amount of target nucleic acid needed to obtain a detectablesignal is about 95% or less of the amount of target nucleic acidrequired in an untagged assay; decreases sensitivity for a targetnucleic acid so that the amount of target nucleic acid needed to obtaina detectable signal is about 105% or more of the amount of targetnucleic acid required in an untagged assay and/or increases replicationprecision by about 105% or more compared to an untagged assay.

The invention further includes a method as described above, wherein thenucleic acid assay is an in vitro isothermal amplification assay.

The invention further includes a method as described above, wherein thenucleic acid assay is an in vitro PCR amplification assay.

The invention further includes a method as described above, wherein thetag is part of an amplification oligomer.

The invention further includes a method wherein the tag is a barcode tagsequence for a sequencing reaction.

The invention further includes a method wherein the tag is a barcode tagsequence for a single molecule sequencing reaction.

The invention further includes a method as described above, wherein thetagged assay decreases the performance parameter by from 25% to 94%,from 50% to 94%, or from 75% to 94% compared to the untagged assay,wherein each range is inclusive of all whole and partial numberstherein.

The invention further includes a method as described above, wherein thetagged assay increases the performance parameter by from 105% to 150%,from 105% to 200%, or from 105% to 500% compared to the untagged assay,wherein each range is inclusive of all whole and partial numberstherein.

The invention further includes a method as described above, wherein thetag sequence has a Tm that is less than or equal to 72° C.

The invention further includes a method as described above, wherein thetag sequence has a primer dimer energy formation that is less than orequal to −10.0 kcal/mol; the tag sequence has a hairpin stability energythat is less than or equal to −4 kcal/mol; the 3′ region of the tagsequence is less than 80% complementary to the one or moreoligonucleotides in the searched database and/or the nucleic acid assaycomprises two or more target nucleic acids.

The invention further includes a method as described above, wherein thedatabase having one or more nucleic acid sequences is a collection ofvarious nucleic acid sequences corresponding to a nucleic acid assay, apublic collection of nucleic acid sequences, an aligned collection ofnucleic acid sequences, the pool of nucleic acid sequences, or acombination thereof.

The invention further includes a method as described above, wherein thedatabase having one or more nucleic acid sequences is a databasecontaining sequence(s) that are derived from: collections of variousnucleic acid sequences corresponding to a nucleic acid assay; a publiccollection of nucleic acid sequences; a collection of aligned sequences,the pool, or a combination thereof.

The present invention also encompasses a nucleic acid tag sequenceobtained by any one of the methods as discussed above.

The present invention also encompasses an amplification oligomer havinga nucleic acid sequence that includes a tag sequence obtained by any oneof the methods discussed above.

The present invention further encompasses a method for identifyingnucleic acid tag sequences for use in an in vitro nucleic acidamplification assay, comprising the steps of: a) generating a pool ofnucleic acid sequences, wherein the pool is at least three nucleic acidsequences from Table 1; b) screening the pool of nucleic acid sequencesagainst a database containing one or more nucleic acid sequences toidentify percent complementarity between nucleic acid sequences in thepool and nucleic acid sequences in the database; c) screening the poolof nucleic acid sequences to determine a performance characteristicselected from the group consisting of: G-C content, multimer formation,primer-dimer formation, Tm, hairpin stabilization energy, self dimerstabilization energy, internal structure formation, G-quartet formation,hybridization energy, activity in an enzyme reaction, and combinationsthereof; d) generating a sub-pool of nucleic acid sequences from theresults obtained in step b), step c) or steps b) and c); and e)selecting one or more nucleic acid sequences from the sub-pool for useas tag sequences in a nucleic acid assay.

The invention further includes a method as described above, furthercomprising: f) synthesizing an amplification oligomer containing a tagsequence selected at step e); and g) performing an in vitro nucleic acidamplification reaction using the amplification oligomer.

The invention further includes a method as described above, wherein thesub-pool at step d) contains nucleic acid sequences with Tm values thatare within ±2 degrees C. from a mean Tm of nucleic acids in thesub-pool; wherein the sub-pool at step d) contains nucleic acidsequences with Tm values that are within ±5 degrees C. from a mean Tm ofnucleic acids in the sub-pool; nucleic acid sequences with Tm valuesthat are within ±10 degrees C. from a mean Tm of nucleic acids in thesub-pool; nucleic acid sequences with G-C contents that are within ±5%from the mean G-C content of the nucleic acids in the sub-pool; nucleicacid sequences with G-C contents that are within ±10% from the mean G-Ccontent of the nucleic acids in the sub-pool; and/or nucleic acidsequences with G-C contents that are within ±30% from the mean G-Ccontent of the nucleic acids in the sub-pool.

The invention further includes a method as described above, wherein thesub-pool at step d) contains nucleic acid sequences with G-C contentsfrom 30% to 80%, from 40% to 70%, or from 30% to 50%.

The invention further includes a method as described above, wherein thesub-pool at step d) consists of the nucleic acid sequences in Table 2.

The invention further includes a method as described above, wherein thesub-pool at step d) contains nucleic acid sequences with lengths from 5nucleobases to 100 nucleobases.

The invention further includes a method as described above, wherein thein vitro amplification reaction performed at step g) has reducedinterference between nucleic acids in the reaction when performed withthe tagged amplification oligomer from step f) compared to whenperformed using an untagged amplification oligomer; the method hasreaction kinetics that are accelerated by about 105% or more whenperformed with the tagged amplification oligomer from step f) comparedto when performed using an untagged amplification oligomer; the methodhas reaction kinetics that are reduced to about 95% or less whenperformed with the tagged amplification oligomer from step f) comparedto when performed using an untagged amplification oligomer; the methodhas increased sensitivity when performed with the tagged amplificationoligomer from step f) compared to when performed using an untaggedamplification oligomer, wherein the in vitro amplification reactionusing the tagged amplification oligomer requires an amount of startingmaterial that is about 95% or less than the minimum amount of startingmaterial required in an untagged assay in order to obtain a detectablesignal; the method has decreased sensitivity when performed with thetagged amplification oligomer from step f) compared to when performedusing an untagged amplification oligomer, wherein the in vitroamplification reaction using the tagged amplification oligomer requiresan amount of starting material that is about 105% or more than theamount of starting material required in an untagged assay in order toobtain a detectable signal; and/or the method has a replicationprecision that is about 105% or better when performed with the taggedamplification oligomer from step f) compared to when performed using anuntagged amplification oligomer.

The invention further includes a method as described above, wherein thetagged assay decreases the performance parameter by from 25% to 94%,from 50% to 94%, or from 75% to 94% compared to the untagged assay,wherein each range is inclusive of all whole and partial numberstherein.

The invention further includes a method as described above, wherein thetagged assay increases the performance parameter by from 105% to 150%,from 105% to 200%, or from 105% to 500% compared to the untagged assay,wherein each range is inclusive of all whole and partial numberstherein.

The invention further includes a method as described above, wherein theone or more nucleic acid sequences in a database is a collection ofvarious nucleic acid sequences corresponding to a nucleic acid assay, apublic collection of nucleic acid sequences, an aligned collection ofnucleic acid sequences, the pool of nucleic acid sequences, or acombination thereof.

The invention further includes a method as described above, wherein theone or more nucleic acid sequences in a database contains sequence(s)that are derived from: collections of various nucleic acid sequencescorresponding to a nucleic acid assay; a public collection of nucleicacid sequences; a collection of aligned sequences, the pool, or acombination thereof.

The invention further includes a method as described above, wherein thein vitro amplification assay is an isothermal amplification assay; amultiplex amplification assay or a PCR amplification reaction.

The invention further encompasses a tagged amplification oligomercontaining a tag sequence obtained by any one of the methods discussedabove.

The invention further encompasses a multiplex in vitro amplificationreaction mixture containing a tagged amplification oligomer with a tagsequence obtained by any one of the methods discussed above.

The invention also encompasses a multiplex in vitro amplificationreaction mixture containing two tagged amplification oligomers, eachwith a tag obtained by any one of the methods discussed above.

The invention also encompasses a multiplex in vitro amplificationreaction mixture, wherein the two tagged amplification oligomers eachhave a tag with the same nucleotide sequence.

The invention also encompasses a multiplex in vitro amplificationreaction mixture containing three or more tagged amplificationoligomers, each with a tag obtained by any one of the methods discussedabove.

The invention also encompasses a kit for amplification of a targetnucleic acid, wherein the kit contains a tagged amplification oligomercontaining a tag sequence obtained by any one of the methods discussedabove.

The invention also encompasses a kit for amplification of a targetnucleic acid, wherein the kit contains at least two tagged amplificationoligomers containing, each containing tag sequences obtained by any oneof the methods discussed above.

The invention also encompasses a kit according as discussed above,wherein the tag sequences are the same nucleic acid sequence in eachtagged amplification oligomer.

The invention also encompasses a collection of nucleic acid sequencesuseful as tag sequences for use in a nucleic acid assay, wherein thecollection contains at least two of the sequences in Table 1, Table 2,or Table 3.

In another embodiment, the method can be applied to sequences intendedto be used in uniplex or multiplex assays.

However, in multiplex assays, interference between the multiple targetnucleic acids, from which at least a part of each is intended to beamplified or detected, can cause reduced and inaccurate measurement ofthe amount of the target nucleic acid in the reaction mixture.

To address this issue, the present disclosure provides asemi-quantitative method that allows for effective discriminationbetween the levels of interference among multiplex systems withdifferent (or same) tag sequences. The method compares the performanceof each of the sequences in a uniplex format, against their performancein a duplex format to arrive at a qualitative determination of theutility of a particular set of sequences together in a particular duplexor multiplex reaction. In a further aspect, the tags are rankedsemi-quantitatively in order of their observed interference in themultiplex reaction. This information can be used for the studies whichwill enable a new user to quickly identify and test tag combinations fora new multiplexed amplification system, and ultimately determine animproved reaction mixture for nucleic acid assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Schematic flow charts illustrating a screen foruniversal Tags. FIG. 1B illustrates that the random oligonucleotidesequences from FIG. 1B are preferably screened in uniplex format andthen in duplex format. However, this is non-limiting. The randomoligonucleotide sequences can be screened serially in uniplex thenduplex formats, or concurrently in uniplex and duplex formats, or induplex format without a uniplex screen, or in any arrangement of uniplexand/or multiplex formats.

FIG. 2. In silico screen: Illustrative blast filtered screen of twoexemplary random oligonucleotide sequences. in this illustration, theblast screen was designed to identify 3′-end dissimilarity, therefore,the sequences selected to advance to the next stage was the sequencesthat was most dissimilar at its 3′-end to sequences in the blast. Therandom oligo sequences are represented as the top line in each of theblast alignments.

FIG. 3. In silico screen: this figure illustrates a series of randomlygenerated oligomer sequences coupled with certain selectcharacteristics. The illustrated characteristics are not limiting.Selections are made based upon the characteristics.

FIGS. 4-10. Each illustrates a uniplex in vitro nucleic acidamplification screen of random oligonucleotide sequences used as tags ina non-T7 amplification oligomer (FIGS. 4-6) or as tags in a T7amplification oligomer (FIGS. 7-9). The target analyte in each of theseassays was PCA3, except FIG. 10 illustrates PCA3 and PSA analytes forcomparison. The in vitro nucleic acid assay format used to generate theresults shown in FIGS. 4-6 was a RUt TMA nucleic acid assay. The invitro nucleic acid assay format used to generate the results shown inFIGS. 7-10 was a RUf TMA, which used an amplification oligomer complexin the direct hybridization configuration. In FIG. 7 (continued) bottompanel, a line is drawn from the number 500 to each of the curvesrepresenting 500 copies of analyte in the reaction. In FIG. 9 Set T9panel there the number 10.sup.4 is shown twice, indicating that a coupleof the 10.sup.4 reactions were above a set threshold fluorescence amountand a couple 10.sup.4 reactions and all of the 500 copy reactions wereunder that threshold amount.

FIGS. 11-17. Each figure illustrates a uniplex in vitro nucleic acidamplification screen using random oligonucleotide sequences as tags innon-T7 and T7 amplification oligomers. Both amplification oligomerspecies (non-T7 and T7) contain a tag sequence. The target analyte wasPCA3. The in vitro nucleic acid assays are TMA reaction in the RUt(FIGS. 11-15) or RUf formats (FIGS. 16-17), with RUf format using adirect hybridization amplification oligomer complex (cPRO). In FIG. 14for the panel showing N28 & T18 tag sequences there are lines drawn fromthe 10.sup.4 copy number to the tracings representing 10.sup.4reactions, and from the 500 copy number to the tracings representing 500copy reactions. IN FIG. 15 for the panel showing the N47 & T9 tagsequences, there is a line drawn from the 500 copy number to thetracings representing the 500 copy reactions.

FIGS. 18-19 show emergence times from a RUf TMA amplification assayusing the T21 and U20 tags incorporated into amplification oligomers ina direct-hybridization amplification oligomer complex. The T21 tag andmodified versions of the T21 tag were used, wherein the modified tagsare shortened by the number of nucleotides in the tag name (e.g. T21-#is shortened by # nucleotide residues). Residues were removed from the3′ end of the tag sequence. The target nucleic acid is PCA3 and isprovided in the assay reactions in 10.sup.4 or 10.sup.2 copy number.

FIGS. 20-21 illustrate uniplex RUh TMA assays wherein the Non-T7amplification oligomers include a tag sequence indicated in each panel.Target nucleic acids are PCA3 (FIG. 20) or PSA (FIG. 21).

FIGS. 22-23 illustrate TMA amplification reactions wherein a reaction isperformed in the presence or absence of a potentially interferingnucleic acid containing a tag sequence. In both of FIGS. 22 and 23, thetop two panels show amplification of PCA3 or PSA using an amplificationoligomer tagged as indicated. No potentially interfering tagged nucleicacid was present. In both of FIGS. 22 and 23, the bottom two panels showa similar amplification as the corresponding top panels, except thatamplification was performed in the presence of a potentially interferingtagged nucleic acid. The potentially interfering tagged nucleic acidused in a duplex oligo reaction was the tagged amplification oligomersdisclosed in the figure, but not directed to the target (e.g., FIG. 22bottom left panel=PSA(U20); FIG. 22 bottom right panel=PCA3(N23); FIG.23 bottom left panel=PSA(U20); FIG. 23 bottom right panel=PCA3(N23)).“NTC” means non-template control and represents a control nucleic acidthat is not targeted by uniplex amplification oligomers or thepotentially interfering amplification oligomer.

FIG. 24 illustrates in the top two panels a TMA amplification reactionperformed in the presence of a potentially interfering tagged nucleicacid (e.g., left top panel is a PCA3 amplification reaction using aU20-tagged non-T7 amplification oligomer in the presence of a U20 taggednon-T7 targeting PSA; and the top right panel is a PSA amplificationreaction using a U20-tagged non-T7 amplification oligomer in thepresence of a U20 tagged non-T7 targeting PCA3). The bottom two panelsillustrate duplex TMA reactions wherein two amplification oligomer setsare provided in each reaction; one directed to PCA3 and one to PSA, andwherein each target analyte is present in the reaction. In these duplexreactions, the amplification oligomer sets each had tagged non-T7amplification oligomers and each used the U20 tag sequence. Theconcentration of target was 10.sup.6 PSA and 10.sup.3 PCA3 for thebottom left panel, and 10.sup.3 PSA and 10.sup.6 PCA3 for the bottomright panel.

FIGS. 25-26 illustrate a semi-quantitative analysis for determininginterference, which can be caused by any of a number of components inthe amplification system. Lower interference values indicate that thetagged nucleic acid used in that system performed better in that systemthan did other tags. Top panels represent a duplex oligo reaction asdescribed for FIG. 22-23 bottom panels. Bottom panels represent amultiplex amplification reaction as described for FIG. 24 bottom panel.The emergence time for each reaction condition to reach 10,000fluorescent units is determined, and then an interference value(1-value) is calculated for each as the sum of the difference betweenthe duplex oligo condition and the corresponding multiplex condition. InFIG. 25, the tagged nucleic acids are the non-T7 amplification oligomersand are both U20 tags. In FIG. 26, the tagged nucleic acids are thenon-T7 amplification oligomers, with the PCA3 non-T7 being tagged withN54 and PSA being tagged with U20.

FIG. 27 illustrates a number of values obtained for tagged amplificationoligomers used in a series of TMA assay as described in FIGS. 22-26.

FIGS. 28 and 30 illustrate the target capture oligomers, blockeroligomers, amplification oligomers and torch detection probes used an aseries of triplex amplification reaction containing varied amounts ofanalytes as indicated in the figures. The top set of oligomer in FIG. 28targets PCA3, the middle set target PSA and the bottom set target aninternal control sequence. In a first set of reactions, the oligomers inFIG. 28 were used against analytes in amounts also as indicated in FIG.28, and the amplification oligomers all used U20 tag sequences. In asecond set of reactions, a set of oligomers substantially identical tothose shown in FIG. 28, except that the U20 tag sequence in the non-T7targeting PCA3, was substituted with an N54 tag sequence. Exemplaryresults for the full U20 tagged reactions and for the U20/N54 taggedreactions are illustrated in FIGS. 29 and 31.

FIGS. 29 and 31 illustrates reaction curves for amplification reactionsusing amplification oligomers having one of the tag sequences selectedaccording to the reactions illustrated in FIGS. 28 and 30.

FIG. 32. T2 ERGa: Comparison of standard u20 with N42 Tag (RUh TMA)

FIG. 33. Triplex RUh TMA reaction containing T2 ERGa/PSA/InternalControl, wherein the all non-T7 amplification oligomers contain a N42Tag.

FIG. 34 illustrates quantitation data from the T2 ERGa/PSA/IC triplexRUh TMA reaction using N42 tagged non-T7 amplification oligomers fromFIG. 33. “Cal” means calibrator and “CON” means control sample.

FIG. 35. Triplex RUh TMA reaction containing T2 ERGa/PSA/InternalControl, wherein the non-T7 amplification oligomers contain N42/N6/N42,respectively.

FIG. 36 illustrates quantitation data from the T2 ERGa/PSA/IC triplexRUh TMA reaction using the N42/N6/N42tagged non-T7 amplificationoligomers from FIG. 35. “Cal” means calibrator and “CON” means controlsample.

FIG. 37. Triplex RUh TMA reaction containing T2 ERGa/PSA/InternalControl, wherein the non-T7 amplification oligomers contain N42/N15/N42,respectively.

FIG. 38. T2 ERGa/PSA/IC (N42/N6/N42) Quantitation

DETAILED DESCRIPTION

In nucleic acid assays which use tags, the selection of the right tag orcombination of tags is important. The presently disclosed methods can beapplied to various nucleic acid assays, but are particularly referencedin regard to nucleic acid amplification and sequencing. However, suchreference is not intended to limit the scope of the application of thedisclosed methods and sequences in any way.

Definitions

Unless otherwise described, scientific and technical terms used hereinhave the same meaning as commonly understood by those skilled in the artof molecular biology based on technical literature, e.g., Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), or other well known technicalpublications related to molecular biology. Unless otherwise described,techniques employed or contemplated herein are standard methods wellknown in the art of molecular biology. To aid in understanding aspectsof the disclosed methods and compositions, some terms are described inmore detail or illustrated by embodiments described herein.

“Activity in an Enzyme Reaction” is used herein to refer to a number ofenzyme driven functions. The term includes binding, extension, cleavage,recombination, repair, and transcription, when these functions areperformed by an enzyme.

“Amplification” of a nucleic acid as used herein refers to the processof creating in vitro nucleic acid sequences that are substantiallyidentical or substantially complementary to a complete or portion of atarget nucleic acid sequence. The in vitro created nucleic acidsequences are referred to as “amplification product” or “amplicon” andmay include one or more tag sequences or the complement of one or moretag sequences. The tag sequences are incorporated into amplificationproduct using tagged amplification oligomers. Alternatively, the tagscan be chemically incorporated into a nucleic acid sequence.

“Amplicon” or the term “Amplification Product” as used herein refers tothe nucleic acid molecule generated during an amplification procedurethat is complementary or homologous to a target sequence containedwithin a target nucleic acid. These terms can be used to refer to asingle strand amplification product, a double strand amplificationproduct or one of the strands of a double strand amplification product.Using an amplification oligomer comprising a target hybridizing sequenceand at least one other region that incorporates into the extensionproduct, results in an amplification product comprising the nucleic acidsequence that is homologous and/or complementary to the amplifiedportion of the target nucleic acid sequence and the incorporated regionsof the amplification oligomer. Incorporated regions forming part of anamplification product include tag sequences.

“Amplification Oligomer” or “Amplification Oligonucleotide” as usedherein refers to a nucleic acid oligomer that is used for generatingcomplementary strands from a target sequence of a target nucleic acid.The complementary strand can be made by elongation of 3′-end of anamplification oligomer, as is common when using primers, or can serve asa recognition site for an enzyme to initiate complementary strandsynthesis, as is common when using promoter sequences. Amplificationoligomers include primers, promoter-based amplification oligomers,promoter primers (which allow for elongation for their 3′-ends andtranscription from their promoter sequences), and promoter-providers(which are modified to prevent elongation of their 3′-ends but allow forpromoter-driven transcription). Amplification oligomers as describedherein may further comprise tag sequences.

Amplification oligomers may be directly or indirectly joined one toanother to form an “Amplification Oligomer Complex.” Typically joinedtogether are first and second amplification oligomers targeting oppositebinding sites of a target sequence. In this configuration, the firstamplification oligomer of the complex hybridizes to a binding site on atarget sequence, while the second amplification oligomer does nothybridize to the target sequence. The amplification oligomers may bejoined one to the other using a connecting compound such as a C9 linker,an oligonucleotide that hybridizes to portions of each amplificationoligomer or other such manners. Alternatively, the amplificationoligomers may be joined one to the other by hybridizing togethercomplementary portions of the amplification oligomers. Amplificationoligomer complexes can comprise any combination of primer or promoterbased amplification oligomer. See US Pat. Pub. No. 2008-0305482 A1 andU.S. patent application Ser. No. 12/828,676 disclosing exemplaryAmplification Oligomer Complexes.

The term “barcode” is used herein to refer to a tag sequenceincorporated into a nucleic acid allowing for identification of somefeature of the nucleic acid. A feature of the nucleic acid includesSNPs. For example, the amplicon species in a SNP analysis aresubstantially identical except for the SNP. Two separate species ofprimers can be configured to have a 3′ end that is complementary to oneSNP sequence or the other, and to have a unique barcode tag sequence toidentify to which SNP species the amplicon corresponds. Detection of theSNP species can then be made by identifying the corresponding barcode. Afeature of a nucleic acid includes the sample from which the nucleicacid originated. For example, a plurality of samples can be analyzed forthe presence of a target nucleic acid. Each sample can be independentlybarcoded by performing an amplification reaction to integrate a barcodetag into the sample target nucleic acid. The samples are then combinedand subjected to subsequent analysis, which can be an additionalamplification or a detection reaction. The various combined amplicons inthe reaction will be substantially identical except for the barcode tagsequences indicating from which sample the amplicon originated. Otherfeatures of a nucleic acid can be identified by a barcode, as isunderstood by ordinarily skilled artisans.

“Complementarity” is the percentage or amount of a sequence that iscomplementary to a target sequence or other sequence. The presentmethods implicate different situations where both high and low levels ofcomplementarity are useful and desirable “Minimal complementarity” asused in this application refers to the desire to achieve the lowestpossible amount of complementarity between a sequence (a tag sequence,for example) and other nucleic acid sequences that may be present in areaction mixture so as to minimize binding/reaction there between. Oneexample is that a tag sequence selected for use in an amplificationreaction is selected to minimize its complementarity to other nucleicacids that may be present in the reaction that are not intended to bindto the tag sequence. In nucleic acid assays wherein a tag sequence isselected so that it is not used to generate amplification products, thenit may be sufficient for only the 3′-portion of the tag sequence topossess minimal complementarity to other nucleic acid sequence and stillfunction as desired. This could be particularly true in the case ofprimers, where the 3′-portion of the sequence is important for enzymaticextension. The 3′-portion of a sequence may refer to the 3′ half, the 3′third or the 3′ quarter of the sequence, or even less as long as theproper function of the tagged oligonucleotide is not prevented orimpeded. In nucleic acid assays wherein it is desired that a tagsequence not randomly hybridize to a nucleic acid in the reaction, thenthe entire tag sequence is screened for complementarity to a nucleicacid in the system.

“Consisting essentially of” is used to mean that additionalcomponent(s), composition(s) or method step(s) that do not materiallychange the basic and novel characteristics of the methods andcompositions described herein may be included in those methods andcompositions. Such characteristics include the method of identifying andselecting nucleic acid tags for use in an assay wherein the tags areselected to reduce or eliminate undesired nucleic acid interactionswithin an assay.

“Database of Nucleic Acid Sequences” is used to refer to an in silicocollection of nucleic acid sequences. The database of nucleic acidsequences can be the pool or sub-pool of tag sequences, or can be acollection of nucleic acid sequences that may be present in a nucleicacid assay. For example, the various nucleic acids corresponding to anamplification reaction to determine whether a blood sample contains HIVcan include the amplification oligomers, the target capture oligomers,probe oligomers, positive control oligomers, HIV nucleic acids and bloodcell nucleic acids. A database of nucleic acids, then, can be any one orcombination of these nucleic acids in the assay system. The database ofnucleic acid sequences can be a public collection of nucleic acidsequences, such as the collection kept by the National Institute ofHealth (GenBank, National Center for Biotechnology Information, U.S.National Library of Medicine, Maryland, USA).

“Dynamic Range” of an assay generally refers to how much a targetconcentration (e.g. in a sample) can vary and still be deleted andquantified. The smaller the range, the less robust is the assay,sometimes measured by the cycle number vs. log of the measured signal.These layer the dynamic range, the greater is the ability of the assayto detect samples/targets with high and low copy number in the same run.

“Hybridization Energy” is used to refer to a measurement of free energyreleased during hybridization of two nucleic acid sequences. Primerdimer hybridization, G-quartet hybridization, primer binding, hairpinstructure formation, internal structure formation, and nucleic acidprobe binding are all examples of hybridizations. Candidate nucleic acidtag sequences are screened for hybridization to determine thesuitability of the tag sequence fro a given application in a nucleicacid assay. In some instances, hybridization energy favoringhybridization is preferred, in other instances; hybridization energydisfavoring hybridization is preferred. For example, in a nucleic acidamplification assay wherein the tag sequences are screened for use aspart of an amplification oligomer, then the hybridization energy foruseful tag sequences includes those that do not hybridize to nucleicacid sequences in the amplification system.

“Interference” during nucleic acid assays is a common recognized problemand can be seen in various aspects of an assay, including targetsequence interference, amplicon interference and primer interference.For example, multiplex PCR assays are known to suffer from primerinterference and dimer formation that can cause a reduction in PCRamplification efficiency and multiplexity capacity. Such interferenceissues can be measured by techniques known in the art.

“Isolated” as used herein means that a target nucleic acid is taken fromits natural milieu, but the term does not connote any degree ofpurification.

“Label” refers to a molecular moiety or compound that can be detected orlead to a detectable response, which may be joined directly orindirectly to a nucleic acid probe. Methods of synthesizing labels,attaching labels to nucleic acids, and detecting labels are well known(e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd ed.(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),Chapt. 10; U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174,and 4,581,333). More than one label, and more than one type of label,may be present on a particular probe, or detection may use a mixture ofprobes in which each probe is labeled with a compound that produces adetectable signal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

“Limit of Detection” or “Detection Sensitivity” generally refer to theability of an assay to detect, measure or otherwise test or react withone particular target or objective as distinctive from others, e.g. in asample. “Limit of Detection” as used herein is a measure of sensitivityof an assay. The detection limit for an analytical procedure can bedefined as “the minimum single result which, with a stated probability,can be distinguished from a suitable blank value” and “the point where,with a stated probability, one can be confident that the signal due tothe measurement can be distinguished from the instrumental backgroundsignal”. For a specific analytical procedure, the LOD can also bedefined as “the lowest amount of an analyte in a sample which can bedetected but not necessarily quantified as an exact value.” The LOD isgenerally expressed as the amount of analyte at which the analyticalmethod detects the presence of the analyte at least 95% of the time. TheLOD is often used in terms of the level at which detection starts tobecome problematic. There are a number of potential reasons for this,inclusive of the presence of noise or an unstable baseline, thecontribution of interferences to the signal, the affect of analyticalblanks, and losses during the extraction, isolation or cleanup process.One of the most important reasons for defining a LOD is to identifywhere the method performance becomes insufficient for acceptabledetection of the target analyte, in order that subsequent analyticalmeasurements can stay away from this problematic area. The evaluation ofthe LOD of an assay is thus critical for trace detection methods,especially where the result will be used for regulatory or public healthapplications.

“Melting Temperature” as used herein refers to the temperature at whichhalf of the DNA strands are in the double helical state and half of thestrands are in a random coil state. The desired melting temperature of acandidate tag sequence varies depending upon the intended use of the tagand the assay format it will be used in. (See e.g., Donald Voet andJudith G. Voet, Biochemistry (1990); Owczarzy et al., Predictingsequence-dependent melting stability of short duplex DNA oligomers,Biopolymers (1997) 44 (3):217-239; and Breslauer, et al., Predicting DNADuplex Stability from the Base Sequence. Proc. Natl. Acad. Sci. USA.(1986) 83 (11): 3746-3750.)

“Nucleic acid” as used herein refers to a polynucleotide compound, whichincludes oligonucleotides, comprising nucleosides or nucleoside analogsthat have nitrogenous heterocyclic bases or base analogs, covalentlylinked by standard phosphodiester bonds or other linkages. Nucleic acidsinclude RNA, DNA, chimeric DNA-RNA polymers or analogs thereof. In anucleic acid, the backbone may be made up of a variety of linkages,including one or more of sugar-phosphodiester linkages, peptide-nucleicacid (PNA) linkages (PCT No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties in anucleic acid may be ribose, deoxyribose, or similar compounds withsubstitutions, e.g., 2′ methoxy and 2′ halide (e.g., 2′-F)substitutions. Nitrogenous bases may be conventional bases (A, G, C, T,U), analogs thereof (e.g., inosine; The Biochemistry of the NucleicAcids 5-36,Adams et al., ed., 11^(th) ed., 1992), derivatives of purineor pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidines or purines withaltered or replacement substituent groups at any of a variety ofchemical positions, e.g., 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, orpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine (e.g. U.S. Pat. Nos. 5,378,825, 6,949,367 andPCT No. WO 93/13121). Nucleic acids may include “abasic” positions inwhich the backbone does not have a nitrogenous base at one or morelocations (U.S. Pat. No. 5,585,481, Arnold et al.), e.g., one or moreabasic positions may form a linker region that joins separateoligonucleotide sequences together. A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in conventional RNAand DNA, or may include conventional components and substitutions (e.g.,conventional bases linked by a 2′ methoxy backbone, or a polymercontaining a mixture of conventional bases and one or more analogs). Theterm includes “locked nucleic acids” (LNA), which contain one or moreLNA nucleotide monomers with a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinity forcomplementary sequences in ssRNA, ssDNA, or dsDNA (Vester et al., 2004,Biochemistry 43(42): 13233-41).

“Oligonucleotide” and “Oligomer” are interchangeable terms and usedherein refer to nucleic acid polymers generally made of less than 1,000nucleotides (nt), including those in a size range from about 5-200nucleotides in length those having lower limit of about 2 to 5 nt and anupper limit of about 500 to 900 nt, or a lower limit of 5 to 15 nt andan upper limit of 50 to 500 nt, or a 10 to 20 nt lower limit and a 25 to150 nt upper limit Preferred oligonucleotides are made synthetically byusing any well known in vitro chemical or enzymatic method, and may bepurified after synthesis by using standard methods, e.g.,high-performance liquid chromatography (HPLC).

“Performance Characteristic” means a characteristic of a nucleic acidtag sequence. A performance characteristic of a nucleic acid tagsequence includes a characteristic that is unique to the tag sequence byitself. This includes, but is not limited to, the length of a tagsequence, the G-C content of the tag sequence, the nucleobasecomposition of the tag sequence, the melt temperature of the tagsequence, etc. A performance characteristic of a nucleic acid tagsequence also includes a characteristic of an oligonucleotide sequencecontaining that tag. A performance characteristic can be independent ofa nucleic acid assay, or a performance characteristic can be determinedwith regard to a nucleic acid assay. For example, a performancecharacteristic that is a hybridization event can be determined for anucleic acid assay, wherein the hybridization is the formation of aprimer dimer. Determination of performance characteristics can be insilico or it can be through wet chemistry.

“Pool of Nucleic Acid Sequences” or “Pool of Nucleic Acids” is used torefer to a collection of nucleic acid tag sequences that will besubjected to an analysis to determine at least one performancecharacteristic. The pool will have at least three nucleic acids. Thepool can be an in silico collection of tag sequences, such as a databaseof nucleic acid tag sequences. The pool can also be a collection of thechemical compounds, such as a freezer box containing a plurality ofvials each with a different tag sequence within. The pool can optionallyinclude other information associated with the individual tag sequences,such as a reference name or a property of the chemical compound.

“Precision” in relation to assays, like PCR, generally refers to thevariability (i.e. the lack of) among repeated measurements orobservations. Precision can be affected by many different factors.

“Precision of Replicates” refers generally to the ability of the assayto produce the same replicates in the same quantities, oftenstatistically measured by means of standard deviations, such as at test.

“Primer” or “Non-Promoter Primer” is an amplification oligomer that doesnot comprise a promoter sequence. Thus, primers comprise at least atarget hybridizing sequence that is configured to be substantiallycomplementary to part of a target sequence. The target hybridizingsequence need not have 100% complementarity to its intended bindingsite, but instead may contain 1 or more of a mismatch, insertion,deletion or modification relative to its binding site, so long as theprimer's target hybridizing sequence hybridizes to its binding siteunder stringent conditions. Primers may further comprise tag sequences,capture sequences, self-complementary sequences for forming hairpinloops, and other sequences in addition to the target hybridizingsequences.

“Probe,” “Detection Probe” or “Detection Oligonucleotide” as used hereinrefers to a nucleic acid oligomer that hybridizes specifically to atarget sequence in a nucleic acid, or in an amplified nucleic acid,under conditions that promote hybridization to allow detection of thetarget sequence or amplified nucleic acid. Detection may either bedirect (e.g., a probe hybridized directly to its target sequence) orindirect (e.g., biotin/streptavidin reporter). Probes may be DNA, RNA,analogs thereof or combinations thereof and they may be labeled orunlabeled. A probe may comprise target-specific sequences and othersequences that contribute to the three-dimensional conformation of theprobe (e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542;6,534,274; and 6,361,945; and US Pub. No. 20060068417).

“Promoter Primer” is an amplification oligomer that is similar to aprimer except that the oligomer further comprises a promoter sequence.Promoter primers are capable of 3′ extension by a polymerase and supplya promoter sequence for transcription by a polymerase. Preferredpromoter sequences include promoter sequences recognized by RNAPolymerases, such as sp6 promoters, T3 promoters, T7 promoters andothers. For example, the T7 promoter sequence is5′-aatttaatacgactcactatagggaga. Promoter-primers, therefore, comprise atarget hybridizing sequence joined at its 5′-end to a promoter sequence.Promoter primers may further comprise tag sequences, capture sequences,self-complementary sequences for forming hairpin loops and othersequences. These additional sequences may be joined to the 5′-end of thepromoter sequence, or they may be joined to the 3′-end of the promotersequence, thereby being flanked on each end by the promoter sequence andthe target hybridizing sequence.

“Promoter Provider” is an amplification oligomer that is similar to apromoter primer except that the 3′-end of the oligomer is modified toprevent elongation by a polymerase. Promoter providers supply a promotersequence for transcription by a polymerase. Promoter providers mayfurther comprise tag sequences, capture sequences, self-complementarysequences for forming hairpin loops and other sequences. Theseadditional sequences may be joined to the 5′-end of the promotersequence, or they may be joined to the 3′-end of the promoter sequence,thereby being flanked on each end by the promoter sequence and thetarget hybridizing sequence.

“Quantization” or “Quantification” is used when referring to accuracy inquantification, precision in quantification, and limit ofquantification. Quantification can be end point quantification.

“Speed of Reaction” as used herein can encompass a variety of reactioncharacteristics by a variety of factors, including the reporter dye,nucleotide and primer concentration, enzymatic activity, and the like(see e.g. Lui and Saint, Analytical Biochemistry 302, 52-59 (2002)).Various mathematical models exist that can describe the reactionkinetics. (see e.g. King et. al., Bio Techniques 47:941-949 (November2009)).

“Region” as used herein refers to a portion of a nucleic acid whereinsaid portion is smaller than the entire nucleic acid. For example, whenthe nucleic acid in reference is an oligonucleotide promoter provider,the term “region” may be used refer to the smaller promoter portion ofthe entire oligonucleotide. Similarly, and as example only, when thenucleic acid is a target nucleic acid, the term “region” may be used torefer to a smaller area of the nucleic acid, such as the targetsequence, an oligomer binding sequence within the target sequence, orthe like.

“Relative fluorescence unit” (“RFU”) is an arbitrary unit of measurementof fluorescence intensity. RFU varies with the characteristics of thedetection means used for the measurement.

“Sequencing” as used herein refers to methods for determining theprecise nucleotide sequence of a target nucleic acid. Various sequencingmethods are known including chain termination sequencing, dye terminatorsequencing, sequencing by ligation, sequencing by synthesis, sequencingby hybridization, circular consensus sequencing, and single moleculesequencing. So-called next generation and third generation sequencingmethods are designed to sequence numerous target templates in parallel.Such methods are particularly useful when the target nucleic acid is aheterogeneous mixture of variants, such as is often the case in a samplefrom a patient infected with a virus, such as HIV or HCV wherein thepatient's viral load typically is a mixed population of a majorityspecies and numerous minority species. Amongst the many advantages,sequencing variants in parallel provides a profile of drug resistantmutations in the sample, even drug mutations present in relatively minorproportions within the sample.

Some next generation sequence methods amplify by emulsion PCR. A targetnucleic acid immobilized to beads via a capture probe provides asuitable starting material for emulsion PCR. The beads are mixed withPCR reagents and emulsion oil to create individual micro reactorscontaining single beads (Margulies et al., Nature. 15 Sep. 2005; 437(7057):376-80). The emulsion is then broken and the individual beadswith amplified DNA are sequenced. The sequencing can be pyrosequencingperformed for example using a Roche 454 GS FLX sequencer (454 LifeSciences, Branford, Conn. 06405). Alternatively, sequencing can beligation/detection performed for example using an ABI SOLiD SequencingSystem (Life Technologies, Carlsbad, Calif. 92008). In anothervariation, target nucleic acids are eluted from the capture probe andimmobilized in different locations on an array (e.g., the HiScanSQ(Illumina, San Diego, Calif. 92121)). The target nucleic acids areamplified by bridge amplification and sequenced by template-directedincorporation of labeled nucleotides in an array format (Illumina) Inanother approach, target nucleic acids are eluted from the capture probeand single molecules are analyzed by detecting in real-time theincorporation nucleotides by a polymerase. The nucleotides can labelednucleotides that release a signal when incorporated (e.g., PacificBiosciences, Eid et al., Sciences 323 pp. 133-138 (2009)) or unlabelednucleotides, wherein the system measures a chemical change uponincorporation (e.g., Ion Torrent Personal Genome Machine (LifeTechnologies, Carlsbad, Calif. 92008)).

As a non-limiting example of identifying and selecting tags for use in asequencing reaction, the following describes identifying and selectingbarcode tag sequences for amplification and detection of majority andminority HIV sequences in a single sample. Human Immunodeficiency Virus(HIV) typically exists in a sample as both majority and minorityspecies. Minority species are often undetected in a sample because oftheir low prevalence (e.g., 0.5% of total HIV population in the sample).Even with assays that are sensitive enough to detect the minorityspecies, the generic nature of many assays hinders resolution of thevarious species (e.g., a PCR assay may detect but not differentiatebetween the species). Minority species are often drug resistant species.A failure to detect the minority species is then a failure to identifyan important component of the tested sample. Retroviral therapy is thenselected and administered without knowledge of the drug resistantspecies, thereby selecting for a drug resistant HIV population in thepatient.

Sequencing assays, including single molecule sequencing assays, areuseful analysis techniques for identifying both the majority and theminority species in a sample because the sequence analysis will providea population of sequence results representing the majority species and apopulation of sequence results representing the minority species. Oftenin sequencing assays, though, the minority species are masked bysequencing errors, which are common with these types of assays.

In order to overcome the error rate problem encountered in sequencingassays and accurately identify minority HIV species in a sample, one candesign a barcoded amplification oligomer that is configured to amplifythe majority species and a separate barcoded amplification oligomer thatis configured to amplify the minority species. The majority HIV speciesbarcoded amplification oligomer can include a 3′ nucleic acid residuethat is complementary to and hybridizes with the SNP nucleobase that isassociated with the majority species. The minority HIV species barcodedamplification oligomer can include a 3′ nucleic acid residue that iscomplementary to and hybridizes with the SNP nucleobase that isassociated with the minority species. The barcode sequence of themajority species amplification oligomer is unique when compared to thebarcode sequence of the minority species amplification oligomer.Furthermore, these barcode sequences are selected using a performancecharacteristic profile that includes providing a unique identifierdespite the error rate associated with single molecule sequencing.Typically, but not necessarily or exclusively, this performancecharacteristic is length and/or nucleobase composition. The lengthand/or nucleobase composition of the barcodes are preferably configuredwith the error rate of the sequencing platform in mind, thereby beingable to buffer any errors from masking the presence and uniqueness ofthe barcode sequences. The majority amplification oligomer and theminority amplification oligomer are then used with a common reverseamplification oligomer, and an amplification reaction can be performed.The amplicons generated from the reaction will include one of the twobarcode tag sequences, depending on from which HIV species the ampliconwas derived. The amplicons can then be sequenced and the data analyzed.Sequencing errors will likely be present and mask the SNP site; however,the unique barcode sequences that were incorporated into the ampliconsbased upon the HIV species from which they derived will provide anidentification of the SNP feature for species. Thus, either or both ofthe barcode tag sequences and SNP residue are good endpoints fordetection of the HIV species in the tested sample.

In the tag identification method for this example, barcode tags would beselected for use in amplification oligomers that are configured togenerate an amplification product from one or another SNP correspondingto different species of HIV. A pool of nucleic acid sequences would begenerated and that pool screened for two or more performancecharacteristics useful for the HIV sequencing reaction. Preferably, butnot necessarily or exclusively, the performance characteristics includelength and/or nucleobase composition. At least one barcode tag sequencewould then be selected for each of the amplification oligomers (i.e.,the majority species amplification oligomer and the minority speciesamplification oligomer) and the barcode tagged amplification oligomerswould be synthesized. One or more performance characteristics can thenbe measured for the various combinations of barcode tagged amplificationoligomers. Two or more barcode tagged amplification oligomers can beused in a sequencing assay. Sequencing data can then be analyzed and thepresence or absence of various HIV species in a sample can be determinedby the sequence data including the unique barcode sequences. Further,the relative abundance of one species to another can be determined bythe sequence data including using the relative abundance of uniquebarcode sequences, one to the other.

As a non-limiting example of identifying and selecting tags for use insequencing assays, the following describes identifying and selectingbarcode tag sequences for amplification and detection of nucleic acidsequences from two or more samples to be combined and sequenced in asingle sequencing reaction. It is often desirable to determine via asequencing reaction the presence, absence or composition of a targetnucleic acid in a number of samples. In one such example, the presenceor absence of a T2:ERG fusion can be determined for two or moredifferent patients in a single sequencing reaction using barcodedamplification oligomers. In such an example, a sample from a firstpatient can be amplified to incorporate a barcode sequence. Separately,a sample from a second patient can be amplified to incorporate a barcodesequence that is unique from the barcode used in the first patient'ssample. Following amplification and preparation of the amplicons for asequencing reaction, the samples can be combined and analyzed bysequencing. Resultant sequencing data can then be identified via thebarcode tag sequences as having come from amplicons of the first sampleor of the second sample.

In the tag identification method for this example, barcode tags would beselected for use in amplification oligomers that are configured togenerate an amplification product of the same target nucleic acid, butfrom separate samples, and then combined for analysis using a sequencingassay. A pool of nucleic acid sequences would be generated and that poolscreened for two or more performance characteristics useful for thecombined sequencing reaction. Preferably, but not necessarily orexclusively, the performance characteristics include length and/ornucleobase composition. At least one barcode tag sequence will beselected for the amplification oligomers used in a sample and at leastone barcode tag sequence that is/are unique from those in the firstsample will be selected for the amplification oligomers used in thesecond sample. The barcode tagged amplification oligomers would besynthesized, and one or more performance characteristics can then bemeasured for the various combinations of barcode tagged amplificationoligomers. Two or more barcode tagged amplification oligomers can beused in a sequencing assay. Sequencing data can then be analyzed for thepresence, absence or composition of the T2:ERG target nucleic acidsequence from each sample by using the sequence data including theunique barcode sequences.

“Sub-Pool of Nucleic Acid Sequences” is used to refer to a collection ofnucleic acid tag sequences that have been subjected to an analysis todetermine at least one performance characteristic, and that are selectedfor incorporation into at least one oligonucleotide sequence for use ina nucleic acid assay. The sub-pool can be an in silico collection of tagsequences, such as a database of nucleic acid sequences. It is notnecessary that the sub-pool is a separate database from the pooldatabase, but instead the sub-pool can be a sub-collection within thelarger pool database that is somehow differentiated. For example, thesub-pool can be a smaller collection of tag sequences within the pool,wherein the smaller collection of tag sequences share a common melt temprange. The sub-pool can also be a collection of the chemical compounds,such as a freezer box containing a plurality of vials each with adifferent tag sequence within. Again, it is not necessary that thesub-pool is physically separate from the pool.

“Tag” as used herein is a user-selected nucleic acid sequence that doesnot hybridize to the target nucleic acid. A tag sequence can be tailoredfor a specific assay against a specific target sequence or can bedesigned to apply to a wide variety of assay formats and/or targetsequences. One or more tags can be used in an assay. When two or moretags are used in an assay, the tag sequences can be different from oneanother or two or more tags in the assay can have substantially the samesequences. Tags can serve a number of functions in nucleic acid-relatedassays, including but not limited to, amplification oligomers, adaptersequences (e.g. SMRTbell), hairpin adapter sequences, sequencingprimers, barcodes, detection sequences, displacer sequences, bindingsite sequences, sequencing primer binding sequence, stem-loop adaptersto circularize a double stranded DNA, capture sequences and the like.Tag sequences may be selected to cause minimal undesired interference inthe assay system using the disclosed methods. Tags are incorporated intoa nucleic acid sequence enzymatically (e.g., using a polymerase toextend a tagged oligomer) or chemically (e.g., using an enzyme freereaction to attach a tag to a nucleic acid). The tags obtained by thepresently disclosed methods may be applied for multiple purposes. Forinstance, in the TMA format, tags can be “joined” to (either directly orindirectly) or incorporated into a primer (or promoter primer, promoterprovider, etc.) to form a tagged primer (or promoter primer, promoterprovider, etc.). When used in an amplification reaction, these taggedamplification oligomers will introduce the tag sequence into anamplification product. Subsequent rounds of amplification can includeamplification oligomers having target hybridizing sequences thathybridize to all or a portion of the incorporated tag. Incorporating tagsequences allows users to control certain aspects of amplification ormultiplex amplification reactions (e.g., primer dimer formation, primerefficiency variations, and other aspects). Tagged amplificationoligomers and amplification reactions are described more fully in UnitedStates Application Publication No. 2008-0305482, the subject matter ofwhich is herein incorporated by reference in its entirety.

“Target Capture Oligomer”, “Capture Oligonucleotide”, or “Capture Probe”refers to a nucleic acid oligomer comprising at least two regions; atarget hybridizing region and a support binding region. The targethybridizing region can be configured to specifically hybridize a targetsequence of a target nucleic acid, or it can be configured tonon-specifically hybridize to numerous nucleic acids in a sample.Specific and non-specific target capture oligomers are described in U.S.Pat. No. 6,116,078 and PCT Publication Number WO 2008/016988. Thesupport binding region is configured to hybridize with a solid support.Preferably, the solid support comprises a complementary binding memberthat binds with the support binding region of the target captureoligomer. These complementary members can be nucleic acids, proteins orother complementary binding members. For example, the support bindingregion can be a nucleic acid and the complementary binding member of thesolid support is a nucleic acid that is substantially complementary tothe support binding region, thereby allowing for hybridization under aset of conditions. An exemplary nucleic acid support binding region is asubstantially homopolymeric tail of about 10 to 40 nucleotides (e.g.,A₁₀ to A₄₀ or, T₃A₁₄ to T₃A₃₀, or T₀₋₃A₁₄₋₄₀. The complementary member,then, is substantially complementary to all or a portion of the nucleicacid support binding region (e.g., A₀₋₃T₁₄₋₄₀. The complementary memberis attached to a solid support, for example, using a covalent linker,ionic interaction, or chelation. Solid supports include nitrocellulose,nylon, glass, polyacrylate, mixed polymers, polystyrene, silane,polypropylene, metal, or other compositions, of which one ismagnetically attractable particles.

“Target Nucleic Acid” as used herein is a nucleic acid on which ananalytical assay is focused. In a diagnostic assay the target nucleicacid is the nucleic acid that, if present in a sample, indicates thepresence of the corresponding nucleic acid of interest or organism. Inan antisense RNA assay, the target nucleic acid is the mRNA that istargeted by the antisense oligomer. Target nucleic acids comprise one ormore target sequences. Target nucleic acids may be DNA or RNA and may beeither single-stranded or double-stranded. The target nucleic acid mayinclude other sequences besides the target sequence. Typical targetnucleic acids include virus genomes, bacterial genomes, fungal genomes,plant genomes, animal genomes, rRNA, miRNA, tRNA, or mRNA from viruses,bacteria or eukaryotic cells, mitochondrial DNA, or chromosomal DNA.

“Target Specific Sequence” or “Target Hybridizing Sequence” as usedherein refers to an oligonucleotide sequence, which is configured tostably hybridize to a portion of the target sequence. In one embodiment,the target specific sequence is fully complementary with the targetsequence, and contains no mismatches. In another embodiment, the targetspecific sequence is complementary to the target sequence and stablyhybridizes to the target sequence under stringent conditions, butcontains 1 or 2 or 3 or 4 or 5 mismatches. In one embodiment, the targetspecific sequence includes at least 10 to as many as 50 nucleotideswhich are complementary to the target sequence.

“Template Sequence” or “Target Sequence” as used herein is a sequencewithin a target nucleic acid on which an analytical assay is focused.For example, if the assay is focused on amplification of a targetnucleic acid, then the target sequence is a part of the target nucleicacid wherein the amplification oligomers are configured to hybridize andgenerate an amplicon. Similarly, if the assay is a sequencing assay,then the target sequence is the portion of the target nucleic acid thatis sequenced. A target sequence can be the entire target nucleic acid orthe target sequence can be a portion of the target nucleic acid. Wherethe target nucleic acid is originally single-stranded, “target sequence”also refers to the sequence complementary to the target sequence aspresent in the target nucleic acid. Where the target nucleic acid isoriginally double-stranded, target sequence refers to both the sense (+)and antisense (−) strands. In choosing a target sequence, the skilledartisan will understand that a sequence should be chosen to distinguishbetween unrelated or closely related target nucleic acids.

Detection of target nucleic acids may be accomplished by using any knownmethod. For example, amplified nucleic acids may be associated with asurface that results in a detectable physical change, e.g., anelectrical change, or can be detected using mass spectrometry. Nucleicacids may be detected in solution phase or by concentrating them in oron a matrix and detecting labels associated with them (e.g., anintercalating agent such as ethidium bromide or cyber green). Nucleicacids may be detected using nucleic acid sequencing techniques. Otherdetection methods use probes complementary to a sequence in an amplifiedproduct and detect the presence of the probe:product complex, or use acomplex of probes to amplify the signal detected from amplified products(e.g., U.S. Pat. Nos. 5,424,413 and 5,451,503, Hogan et al., U.S. Pat.No. 5,849,481, Urdea et al.). Other detection methods use a probe inwhich signal production is linked to the presence of the targetsequence. In some instances, the probe is degraded by the amplificationenzyme to release the fluorophore from the presence of the quencher(e.g., TaqMan, U.S. Pat. No. 5,210,015). In other instances a change insignal results only when the labeled probe binds to amplified product,such as in a molecular beacon, molecular torch, or hybridization switchprobe (e.g., U.S. Pat. Nos. 5,118,801 and 5,312,728, Lizardi et al.,U.S. Pat. Nos. 5,925,517 and 6,150,097, Tyagi et al., U.S. Pat. Nos.6,849,412, 6,835,542, 6,534,274, and 6,361,945, Becker et al., US2006-0068417 A1, Becker et al., and US 2006-0194240 A1, Arnold et al.).Such probes typically use a label (e.g., fluorophore) attached to oneend of the probe and an interacting compound (e.g., quencher) attachedto another location of the probe to inhibit signal production from thelabel when the probe is in one conformation (“closed”) that indicates itis not hybridized to amplified product, but a detectable signal isproduced when the probe is hybridized to the amplified product whichchanges its conformation (to “open”). Detection of a signal fromdirectly or indirectly labeled probes that specifically associate withthe amplified product indicates the presence of the target nucleic acidthat is amplified.

“Transcription Mediated Amplification” refers to an isothermalamplification method wherein at least a portion of the amplificationcycle include making RNA transcripts from a target sequence. dependingon whether the primer or the promoter based amplification oligomer isdesigned to hybridize the initial target nucleic acid in a sample, thenthe TMA assay is referred to as reverse TMA (RTMA) or forward TMA (TMA).When using tag sequences in one or more of the amplification oligomerspecies in the reaction, the reactions are referred to herein asfollows. (a) “Full,” meaning that the amplification oligomer speciesused in the reaction include a first tagged-amplification oligomer in a1:1 ratio with the captured target nucleic acids; a secondtagged-amplification oligomer in a 1:1 ratio with the captured targetnucleic acid; an excess of a first amplification oligomer that targetsthe complement of the tag sequence of the first tagged-amplificationoligomer; and an excess of a second amplification oligomer that targetsthe complement of the tag sequence of the second tagged-amplificationoligomer. A 1:1 ratio of the tagged-amplification oligomers is typicallyaccomplished by including the first tagged-amplification oligomer andthe second tagged amplification oligomer in a target capture reagent asan amplification oligomer complex. Amplification oligomer complexes aredescribed in U.S. Published Application No.: 2011-0003305, and include,but are not limited to, direct-hybridization complexes, covalentlylinked complexes and others. Briefly, a target capture reaction isperformed wherein a target capture oligomer and an amplificationoligomer complex are both hybridized to a target nucleic acid. Thehybridized nucleic acids are then removed from the remaining componentsof a sample, typically using a solid support like a magnetic bead,optionally followed by one or more wash steps. These removed componentsare then added to an amplification reaction mixture that containsreagents for performing the amplification reaction. It is understoodthat for the three-quarters and the half reactions described below, thatthe 1:1 ratios are typically achieved by including just the taggedamplification oligomer in the target capture reaction, thoughamplification oligomer complexes comprising a tagged amplificationoligomer member and a non-tagged amplification oligomer member can beused. (b) “Three-quarters,” meaning that the amplification oligomerspecies used in the reaction include a first tagged-amplificationoligomer in a 1:1 ratio with the captured target nucleic acids; anexcess of second tagged-amplification oligomer; an excess of a firstamplification oligomer that targets the complement of the tag sequenceof the first tagged-amplification oligomer; and an excess of a secondamplification oligomer that targets the complement of the tag sequenceof the second tagged-amplification oligomer. (c) “Half” meaning that theamplification oligomer species used in the reaction include a firsttagged-amplification oligomer in a 1:1 ratio with the captured targetnucleic acids; an excess of a second amplification oligomer; and anexcess of an amplification oligomer that targets the complement of thetag sequence of the first tagged-amplification oligomer. As is usedherein, RUf means Reverse TMA with the full tagged amplificationoligomers format. RUh means Reverse TMA with the half taggedamplification oligomers format. RUt means Reverse TMA with thethree-quarters tagged amplification oligomers format. (See e.g., WO2011/003020, throughout and particularly in the Examples discussinghalf, full, three-quarter and other tag arrangements in TMA reactions;and see WO 2009/140374, throughout and particularly in the figureswherein the flow of a TMA reaction is illustrated. Both of thesedocuments are incorporated herein by reference.)

A. Description of the Method for Identifying and Selecting Tags for Usein In Vitro Nucleic Acid Assays.

The overall method can be schematically represented as is generallyshown in FIG. 1A. The Figures illustrate the general tag identificationmethod by reciting an amplification assay as the in vitro assay fortesting tagged nucleic acids. As is made clear in this disclosure,though, the tag identification methods are applicable to a variety of invitro assays, not just amplification assays. Tags are identified asbeing useful in an in vitro assay that contains additional nucleic acidsequences. Identification is based upon a number of factors, asdescribed herein. Once identified, the tags can be incorporated into oneor more nucleic acids used in the in vitro assay. The steps for tagidentification are as follows:

1. Random Oligo Generation: To begin, a pool of oligonucleotidesequences is obtained or generated (as necessary), typically as a randompool of sequences. This can be performed in a variety of ways, includingwith the aid of a computer program. If desired, the user can setboundaries at this stage for desired characteristics or properties, suchas sequence length and GC content. When a computer is used, this phaseof the method can be described as an in silico screen. Sequence lengthas a parameter or boundary will be set depending upon the desired use ofthe tag. Lengths can be set, for example, to 25-30 nucleotides. G/Ccontent is the amount of G residues and/or C residues in anoligonucleotide or polynucleotide sequence. G/C content in the Tagsequences is reflected as a percentage of the number of G and/or Cresidues as compared to the total number of residues in theoligonucleotide. Among other things, the G/C content alters the meltingtemperature of the sequence. Acceptable G/C content depends on thetarget nucleic acid under consideration. Typical G/C content can be inthe range of 30 to 80%, 40 to 70%, or 30 to 50%.

2. Blast: The pool is subjected to a search to identify sequences thathave minimal sequence complementarity to all known sequences in one ormore selected databases to obtain a sub-pool of sequences. An example ofa useful tool for this purpose is the BLAST (Basic Local AlignmentSearch Tool) algorithm available from NCBI (National Center forBiotechnology Information). Sequences selected for the sub-pool arethose having complementarity of less than 95%, less than 80%, less than70% or less than 50% to the nucleic acid sequence in the database.

3. Screen for Oligonucleotide Parameters: The sub-pool of sequencesprepared from the step 2 Blast is then subjected to further screening,which may be in silico screening, for features that could negativelyaffect their performance in regards to the desired functionality,including melting temperatures, activity in an enzyme reaction, G-Ccontent, hybridization energy, multimer formation, internal structureformat, G-quartet formation and hairpin stability. A variety of featurescan be used for the in silico screening, depending upon the desired useof the tags. For example, if the intended use of candidate sequences isas primers/providers in an amplification reaction, the candidatesequences can be screened for one or more of primer/dimer formation,internal secondary structure, inappropriate melting temperature (Tm)values, cross-reaction or other unwanted interactions with other nucleicacid sequences to be used in an assay (including other tag sequences),and the like. Sequences identified to possess an undue amount ofnegative attributes are removed from the sub-pool. The following areexemplary parameters that can be screened for at this stage.Primer/dimer formation is a characteristic to be avoided in identifyinguseful tags for an in vitro amplification assay. Primer-dimer formationcapability can be determined using software routinely available to oneof ordinary skill in the art, e.g. Oligo Primer Analysis Software fromMolecular Biology Insight, and references therein. Some internalstructures are also understood to typically be undesirable in a nucleicacid assay system, such as the presence of hairpin loops, G-quartets andother unwanted structures. Such structures can be easily identified bymethods known to those skilled in the art. Inappropriate meltingtemperature (Tm) value is another characteristic to be avoided in usefultags. Cross-reaction or other unwanted interactions with other nucleicacid sequences to be used in a reaction mixture (including other tagsequences) are also to be avoided for useful tags. Cross-reactions to beavoided can be from target nucleic acids or other nucleic acidssuspected of being present in a sample and/or assay reaction, such aspathogenic or non-pathogenic organisms, mammalian nucleic acids,contaminating nucleic acids from enzymes, side-products of nucleic acidamplification reactions, etc. Cross-reactions can also occur withsequences intentionally included in the assay mixture. For instancecross reactions can occur between a tag sequence and the targetsequence, the tag sequence and the target specific sequence of anoligomer, and the tag sequence and other templates, targets, primers,probes, detection sequences, displacer sequences, binding sitesequences, capture sequences etc.

4. Synthesis and In Vitro Assays: Based on the results from thescreening steps described above, performance of candidate sequences canbe optimized (if desired) by a cycle of systematic sequence designchanges followed by a repeat of one or more of the in silico screendescribed above for the sub-pool and the in vitro screen discussedabove.

The sequences that successfully pass the above two screens are thenselected for use in the intended nucleic acid assay, or as a componentin a reaction mixture, and subjected to rigorous experimentation tobenchmark their activity against the desired performancecharacteristics. This additional experimentation is generally conductedin vitro, that is, the sequences are synthesized and run in an in vitroassay. Cross reactivity in a system inhibits or considerably degradesthe amplification performance. The practitioner can then systematicallyeliminate tags from the candidate tag pool which cross-react with othertags, amplification oligomers, or particular templates, to obtain anon-biased assay.

In typical multiplex amplification reactions, a variety of undesired“side reactions” could occur that ultimately degrade assay performance.For example, various amplification oligomers can interact with oneanother or with other sequences within the assay. Commonly, primersand/or providers directed towards one target nucleic acid (or group oftarget nucleic acids) interact with primers and/or providers directedtowards another target nucleic acid (or group of target nucleic acids),causing degraded performance of one, or the other or both amplificationsystems. This problem typically gets worse as the number of targetsequences present in a multiplex reaction increases. This problem can bereduced or solved by incorporating a tag sequence into one or moreamplification oligomers in the system. The tagged amplificationoligomer(s) will then incorporate their tag sequences into the initialamplification product. Subsequent amplification can take place usingamplification oligomer comprising sequences that are configured tohybridize to the incorporated tag regions

However, even if specific primer and/or provider interactions arereduced or eliminated, other interactions that degrade assay performancecan still be present. For example, the two tagged primers (or primer andprovider) can interact with one another. In the half taggedamplification mode described above, the one tagged primer (for example)can interact with other remaining specific primers and/or providers.Furthermore, tagged primer(s) can interact with other oligos in theamplification reaction, such as probes, blockers and target captureoligomers (TCOs), and/or with target nucleic acid related sequences(such as amplicons). Additionally, in an amplification system using twoor more tagged amplification oligomers, subsequent amplificationreactions are driven by the same amplification oligomer(s) complementaryto the tag sequence. Competition between the various amplificationreactions for this limiting resource can be a problem. For example,amplification of a target sequence present in a multiplex reaction athigh levels will consume the primers complementary to the tag sequencebefore amplification of a target sequence present in a multiplexreaction at low levels can “complete” its normal reaction.

One solution to this problem is to create unique tag sequences for someor all of the target sequences in a multiplex reaction, and to designthem such that they do not interact with each other, or with any othertag set from different target sequences in the reaction, or with anyother sequences in the reaction mixture. In this way, each amplificationreaction can proceed independently and degradation of multiplexamplification performance is reduced or eliminated. The identificationand selection method of this disclosure can be used to generate suchunique tag sets.

B. Description of the Method for Identifying and Selecting Tags HavingMinimized Interference in a Multiplex Assay.

The present invention further encompasses a method that draws on thepower of combinatorial screen and selection to identify improved tagsequences that reduce or eliminate the problem of interference, which isgenerally observed in multiplexed amplification reactions. Anotheradvantage of the method is that the criteria set for the screen can bevaried to match a desired property that the user wants to screen for ina particular assay system. These criteria can be speed of anamplification reaction of a first target nucleic acid relative to theamplification speed on other target nucleic acids, performance of a tagsequence or tagged oligomer within a give system, lack of interferencewith or by other nucleic acids in the assay system, etc. The finalsolution in terms of tag sequence(s), number of tags to use in a system,which nucleic acids comprise tags, arrangement of tags and etc. isderived from a large repertoire of user-defined oligonucleotide tagsequences (as opposed to target-specific sequences). Thus, the methodtranscends the inherent limitation imposed by amplification systems thatuse target specific primers for amplification. Moreover, the tags areincorporated into the amplicon and can be utilized to enable downstreamapplications such as sequencing, signal amplification, microarrayanalysis, etc. One can envision the selected tags to be modified assignaling probes, adapters for ligation-based assays, as tags insequencing applications and the like.

The overall method can be schematically represented as shown illustratedin FIG. 1B for using tag sequences in a given amplification format. In amultiplex amplification assay, one or more tags are selected by thefollowing steps: 1-4. Preparation of Library of Tag Sequences: A libraryof tag sequences is prepared using the method described above foridentifying useful tags, namely: 1. Random oligo generation; 2. Blast;3. Screen for Oligonucleotide Parameters; 4. Synthesis and in vitroAssays After the completion of these steps, further identification ofuseful tags can be determined using the following steps:

5. Screening in Uniplex: A library of tag sequences is screened for eachtarget sequence individually in assays, for example in a uniplex nucleicacid amplification format, such as TMA. Each tag sequence is thusevaluated qualitatively or quantatively, based on, for example, theprecision between replicates limit of detection, sensitivity, kineticsof reaction and emergence time relative to a standard reference tag, ifavailable, or other appropriate parameters as desired.

6. Screening Using Duplex Oligos: The tag sequences demonstratingoptimal performance from the step 5 uniplex screen are subsequentlyscreened in the presence of oligos of the competing target sequence(duplex oligo screen). This screen is done first without using a tag forthe second target sequence, and then including the tag chosen from Step5 for the second target sequence (this could be the same tag or anotherunique tag) in order to assess the level of oligo interference existingin a given multiplex amplification. The level of oligo interference foreach target sequence is again determined qualitatively or quantitativelyrelative to their performance in uniplex assays.

Methods for such quantitative or qualitative determination are known tothose skilled in the art and include, for example, by comparing Ctvalues between the uniplex and multiplex reactions, by determining thelimit of detection of a particular target nucleic acid in a uniplexreaction and in a multiplex reaction, etc.

7. Screening Using Duplex Oligos and Target Nucleic Acids: Each tagsequence pair showing minimal oligo interference and sufficientperformance in the presence of duplex oligos from step 6 are thenscreened in the presence of duplex oligos and target sequences toascertain the level of total interference. More specifically, relativelylow copy levels of each target sequence are evaluated in the presence ofhigh copies of the competing target sequence (in two separateconditions) with the tag of each target sequence chosen from the duplexoligos screens. The relative amounts of each target sequence to be usedfor these interference screens, where the target sequence interferencecan be observed from the amplification curves might be different foreach system and therefore should be chosen on a case-by-case basis. Theresults of the reactions in steps 6 and 7 are then qualitatively andquantitatively evaluated to determine preferred combinations of tags tobe used in an assay given the nucleic acids present in the multiplexreaction mixture.

Such qualitative evaluation methods are known to those skilled in theart and include determining whether or not one target nucleic acid canbe detected (at a certain input copy level) in the presence of the othertarget nucleic acid(s).

8. Interference Analysis: A novel method to quantitate the interferenceobserved for each combination of tags screened as described above hasbeen developed. With this method, an “interference value”, or “I-value”,is determined for each target sequence in a multiplex reaction, andthese I-values are added together to yield the total I-value. Forexample, in a duplex system, [I-value (total)=I-value (targetsequence1)+I-value (target sequence2)].

When using Real-Time TMA I-values can be calculated from emergencetimes. In a duplex system, for example, the I-value of target nucleicacid 1 is calculated by subtracting the emergence time of a relativelylow copy level of target nucleic acid 1 in the absence of target nucleicacid 2 from the emergence time of the same (low) copy level of targetnucleic acid 1 in the presence of a relatively high copy level of targetnucleic acid 2. In an equivalent manner, the I-value of target nucleicacid 2 is calculated using a relatively low copy level of target nucleicacid 2 in the absence or presence of a relatively high copy level oftarget nucleic acid 1. The sum of these two I-values yields the totalI-value for the given set of tags used. The lower the total I-value, theless interference there is between the tags. FIGS. 25 and 26 illustratethe calculation of I values for amplification assays using different tagin the primers. A wide variety of tags and combinations thereof can bescreened and the relative interference levels can be rapidly quantitatedusing this method.

EXAMPLES

The following examples demonstrate the use of this method to select thebest tag sequences for use in a TMA assay format. These examples areintended only to demonstrate the use of the selection method and are notintended to limit the scope of application to only TMA or to onlyamplification assays.

Example 1 Screen for NT7 Tag Sequences

Step 1—Random Oligo Generation: A large pool of oligonucleotides 25-30nucleotides in length are randomly generated. Random oligonucleotidesequences can be determined in a variety of manual or automated methods.One manual method for determining random oligonucleotide sequences of adesired length includes a blinded selection of a series of A, C, T, Gand/or U residues. One automated method for determining randomoligonucleotide sequences of a desired length includes using analgorithm for randomly selecting a series of A, C, T, G and/or Uresidues. Many algorithms are freely and commercially available togenerate a random pool of nucleotide sequences. Suitable algorithmsinclude those found at http://molbiol.ru/eng/scripts/01_16.html;http://www.faculty.ucr.edu/˜mmaduro/random.htm;http://tandem.bu.edu/rsg.html;http://www2.unijena.de/biologie/mikrobio/tipps/rapd.html; and, describedin Piva and Principato, In Silico Biology, 6, 0024 (2006). Many otheralgorithms can also be used. For this example, an initial population of˜1000 sequences was generated for screening (see step 2 below). Thesesequences were selected to vary in their GC content, length and Tm.

Step 2—Blast: The oligonucleotides generated in step 1 above were thensubjected to an in silico screen using the BLAST (Basic Local AlignmentSearch Tool) algorithm available from NCBI (National Center forBiotechnology Information) website. The basic workflow for eacholigonucleotide was as follows:

Using the “BLAST Program Selection Guide” available on the website,“nucleotide blast” (blastn) was chosen to search the nucleotidedatabases using a nucleotide query. The program “blastn” is specificallydesigned to efficiently find short alignments between very similarsequences and thus is the best tool to use to find the identical matchto a query sequence. In this example, the query sequence was one of thetag sequences identified in step 1. Several different databases areavailable for search using BLAST, and the “nr” nucleotide database wasused in this instance.

The goal of this screen was to identify sequences with minimalcomplementarity to sequences in the data base other than the desiredtarget nucleic acid, thus minimizing the potential of unwanted crossreactivity with non-target nucleic acids that may be present in theassay reaction mixture. Particular care was taken to screen sequencesthat may be problematic in a given assay. For example, if a viral assayis being developed, the sequences corresponding to other non-targetviruses as well as the non-target regions of the targeted virus werecarefully examined.

If a candidate tag sequence yielded greater than about 80% overallcomplementarity to any sequence in the data base, it was rejected. Thevalue for percentage complementarity used as a cut-off will varydepending on the assay under developed as well as the specificrequirements of that assay. The 80% value cited here is only an exampleof a cut-off. Additionally, candidate tag sequences that yielded greaterthan 6 to 8 contiguous bases of exact complementarity were also rejectedin general. However, as discussed above, exact complementarity in the 3′portion of the molecule is undesirable if the tag is to function as aprimer, for example. Therefore, more exact complementarity than listedabove could be tolerated in the 5′ portion of a tag primer candidate inthis case, as long as 3′ complementarity was low. FIG. 2 shows anexample of such an analysis.

Other rejection criteria can also be set, depending on the particularspecifications of the assay under development. Based on the chosencriteria, a fraction of the tag sequence candidates are rejected and theremainder taken on to the next screening step.

Step 3—Screen for Primer Parameters: A pool of about 100 sequences(Table 1) was identified in steps 1 and 2 above out of an initialpopulation of ˜1000 randomly generated sequences. These 100 sequenceswere then subjected to another screen wherein properties that coulddecrease a tag candidate's effectiveness in the desired application,such are hairpin or primer-dimer formation, were identified and thosesequences were removed from the pool. For this second screen, the oligoanalyzer software available from Integrated DNA technologies(www.idtdna.com/analyzer/Applications/OligoAnalyzer) was used.

Representative data from this second screen are shown in FIG. 3.Candidate tag sequences that were predicted to form hairpin structureswith a stability of −4 kcal/mole or greater (i.e., a more negativevalue) were discarded. Candidate tag sequences that were predicted toform primer-dimer structures with a stability of −10 kcal/mole orgreater (i.e., a more negative value) also were discarded. Candidate tagsequences with Tm values >72° C. were also discarded in this example.

Table 1 presents the results of the screens, wherein the asterisk symbol“*” following a value indicates that the corresponding sequence isdiscarded as a candidate tag sequence based on that result. Sequenceswhich lack any asterisk are selected as tags.

TABLE 1 Results of Tag Parameter Screens Hairpin self dimerstabilization stabilization energy energy SEQ maximum maximum Seq IDSequence GC Tm (dG) in (dG) in # NO: (5′-3′) Length content (° C.)KCal/mol) KCal/mol    1   2 CCCCGTCAAACAAAAACGG 30 56.7 65.9 −4.81−11.00* GAGCGTGTACC    4   3 CCATAGGCCTTCTGCACTG 30 53.3 63.2 −1.18−12.47* CTCCATATACC    6   4 GTCCCCATCGGAGGGCATC 30 60 67.5 −3.36 −8.16TTATCGTGCCT    8   5 CCGCCCTCCTTCGCCCCCC 30 66.7 70.1 −1.67 −9.75GGTGAAATAAC   12   6 AATGCTCACCTCTATTCGG 30 46.7 60.9 −0.71 −5.13GACTTGAGTAC   21   7 CCCGCGCACCACCTCCATC 30 66.7 70 −2.19 −10.36*ACGCAGAAGAG   25   8 GTCGGAACGCCAGGTACAG 30 60 66.7 −2.08 −9.89TTAGCGCATCC   26   9 AAGTCACTGGCCAGCATAA 30 53.3 65.4 −0.76 −16.38*TGCGTGAAGGG   27  10 GTGATGCTTTATGAGATTC 30 50 61.7 −2.15 −9.75CGGTCTCCGAC   28  11 GACGGTGCATCACCCGCAT 30 60 67.6 −2.79 −7.05TTGCTGTAGCG   34  12 AGAATTCTTGCAGGTAGAG 30 46.7 62.2 −2.22 −11.71*GTCCCCTCATT   35  13 AAGCCAAAATTACAATCGA 30 40 59.1 1.41 −9.71TCCCTACCAAC   37  14 ATCTTGCACCTTCCCAGAT 30 50 64.3 0.4 −7.05GTAAACCCCCT   42  15 GAAGCGGCAGCTCAGCCGG 30 66.7 69.6 −6.97* −9.82TTCTCGGAGAG   43  16 GCACGCGGGCTCCTTGGGA 30 60 67.1 −0.1 −10.36*CACTATGATTG   61  17 CCCATCAGGACAGTCAGCT 30 56.7 66.5 −1.35 −10.24*GCCCACGAATT   78  18 CTTTAGTGCGGTAGGACCG 30 56.7 64 −5.07 −10.58*AGACTACCGTG   79  19 TTATGTGCCAGCTGGGCCT 30 63.3 69.6 −2.6 −16.38*AAGGCTCCGGG   80  20 GACTCTCCTAGGGCGTTCG 30 63.3 67.3 −0.43 −10.30*TCTGGGACTGC   82  21 CGGAGAATACCCTCGACTG 30 46.7 60.1 −0.83 −6.76TATCATATCGT   84  22 TTCATCGAGGTACATTGGT 30 40 59.6 −0.18 −6.76GCTATTCCATT   86  23 TACCACCTGGTTCAAGGTG 30 60 67.9 −3.73 −10.87*TGCCGTACGCG   87  24 AGGAGAACCAGCCTGGAGC 30 53.3 64.8 −1.93 −6.62GTTTAAGCATC   88  25 GATGTCCTAAAATGAGGCG 30 46.7 60.6 −0.28 −4.67TGGCAATAGAG   89  26 CAGAGTCATGTATACCCAC 30 50 62.1 −0.19 −6.76TGTCGGTCGAA  104  27 GTCAGGCTAGGGGGTTATC 30 63.3 68.2 −2.62 −6.14CCAGCAACGGC  106  28 TGGGTTCTGCTAACCGGTG 30 53.3 65 −2.02 −12.43*CCGTTCTTAAC  116  29 TTTTTGACAGTGATGAAGA 30 43.3 60.7 −1.61 −3.65GGGAGGTACGA  133  30 GAGAACTCGCGCTCCCTCA 30 56.7 65.4 0.21 −10.36*CTCCGTTTAGA  136  31 CTATGGTTCGTTACTGAAT 30 46.7 61 −1.19 −7.13CGAAAAGCCGC  138  32 TAGCTATCAAAACAGGCGT 30 43.3 60 −1.05 −8.26CATCGGTTAAG  145  33 AGGACGCTGACACCGTTGG 30 60 68.1 −4.77* −9.69GGTAAAGCGTG  152  34 CCTGCTTAGGGTCACTTAA 30 53.3 63.5 −0.37 −9.89ACTACTGGCGC  155  35 GGTGATGGCCCATACCGAT 30 66.7 70.1 −1.55 −9.28CACGCCCGCAG  156  36 CGGCAGGAGGGACTGCGAT 30 60 66.5 −2.59 −6.69TTCCATAGAGC  159  37 TGGCCGGAGAGAGGATAGG 30 60 67.5 −1.62 −9.75AAGCGGGACTA  161  38 TAGCAGGTGTCTCGGTCCT 30 53.3 64.6 −4.25* −7.05CAACTGCAAAC  163  39 ACACATCCCAGGACTGCCG 30 60 68.5 −1.8 −9.28TGGCCTACGTA  171  40 GTGCTAGCCCGGGCCCTTC 30 63.3 69 −3.7 −22.17*TTAACTCGGGA  172  41 CGGAATCTGAACATCTATC 30 53.3 64.5 −3.69 −10.36*AGAGCCGCGCT  174  42 GACGAGCTTGTTCCAATTC 30 56.7 65 −2.66 −9.96CTCGAGCCGAG  179  43 GTTGGGGAGGGGCACTACG 30 60 67 −1.96 −3.61ACTTAGGGCTA  182  44 AATGTGGACGGCCGCTCCG 30 56.7 67.3 −3.59 −16.50*TACTTCTGACA  183  45 AGGGCCAGCAGCTGGTTCC 30 60 69.3 −2.32 −10.24*TTCGCCAGTTA  185  46 GGCCGTCAATGTGTTTTGC 30 56.7 67.7 −1.48 −9.75ACCCAACCGGA  188  47 CAGTGACTGGGCTAGTGAA 30 53.3 62.9 −4.43* −7.81GTGAGTCACAG  193  48 TCCCACGTCCTTCGACGCA 30 53.3 65.6 −3.02 −6.76CACTGTAACTT >301  49 TCATGTATCGCCCGTGGGT 25 56 62.3 −0.69 −6.34AAGCTC >305  50 ATGTTATGGAGAGTGGGTT 25 44 57.9 0.75 −3.14 AGGCAA >307 51 ATGAGGGAGTAAGGAGATT 25 44 55.4 0.57 −2.91 AGGTTC >309  52CATGCTGCCCGCATACACT 25 64 66.5 −6.62* −14.84* TGCGGG >313  53GCCCAGCAGTTATACAATT 25 52 60.3 −0.2 −6.21 CGTGGC >314  54TTGGGCTCTCCAGTAGCCG 25 52 62.2 −2.14 −7.81 AACAAA >316  55TGACGTTAAACGCAATCCG 25 44 59.4 −3.94 −10.36* CGTAAA >325  56GTCGCCATTCAGGACACGC 25 56 63.4 −1.92 −10.36* GAAACT >327  57GTGGTTGCTACAGCCTAGC 25 52 60 −1.79 −5.7 CTAGAT >336  58CCACTTTTCATTCCGAGTC 25 56 62 −0.08 −10.36* CACGCG >339  59AGGAGGAACCGGAAGATCT 25 48 57.6 −1.09 −9.75 AATCTG >342  60CCAATGCTTTCAAATAACC 25 40 56.2 0.57 −3.89 CGTTCT >343  61GCGACTGTGGCAACCCCAT 25 60 66 −3.65 −8.33 TTCGCA >346  62AAAAAACGGAGGAGTCGAA 25 48 59.4 −0.75 −6.76 CCTTGG >350  63AGTTGGATGGATATCTCGC 25 48 59.4 −0.32 −7.06 TCGTGA >356  64CGCTGTCCTCTCTGACACT 25 52 60 −1 −4.87 AAAGGT >357  65ATTTCAATAGTCAACCCGG 25 40 56.1 0.44 −9.75 TATCCA >505  66TTCGCGCCAGCGACCCCAC 25 60 66.3 −3.52 −10.36* TTATGA >510  67GGTTGGGGGGCTCGGCTCA 25 64 65.2 0.09 −5.38 TGTATC >516  68ATGATGCTGAATCGCGATG 25 60 65.1 −0.13 −16.46* GGGGGG >517  69TAAGGAGACTAGGTTCCAA 25 44 55.5 −0.84 −6.34 TAGCTG >523  70TTACACAAATCGTGGGTTG 25 48 60.6 −1.86 −9.28 GCCTCT >534  71CGAAAGCGTTCCGCAGGAC 25 64 67.1 −2.4 −6.75 CCCCTT >538  72CACCCTTGGACACGTGGAA 25 64 65.7 −1.49 −10.20* GTGGGC >541  73AAAGTCTGAGAATGAGTGA 25 36 53.8 0.92 −3.43 TACCAT >544  74ATATTGGTAGTTTTGTCCG 25 40 54.9 0.67 −3.91 CTGTAG >549  75CGGAAGATCTAATCTGCAC 25 44 57.5 −0.78 −7.82 GCAATT >608  76GCGCCTCGTTGGGCAGAAG 30 53.3 66 −2.36 −9.89 TTTGTGGAAAT >610  77ATCTTCACCTACCGAGTTC 30 53.3 63.4 −2.39 −9.28 TACGGGCCTAC >613  78CCCACAACTTGCACCCGCT 30 63.3 68.9 −2.32 −7.05 ATGCGACCCTG >618  79GCCCAGGAGCTCTCCTGGG 30 63.3 67.5 −7.98* −15.93 TAACAGTAGCG >620  80CACGGCCCCCAGGCGGCGT 30 70 72.5* −3.3 −9.28 ATCAGGGATGA >623  81TCCCCGGCACGGACCGCAA 30 70 73.2* −5.63* −9.75 GGGACCAAAGC >629  82GATTAGTGGCCCAACGGGA 30 50 63.6 −2.04 −9.28 ACAAACTTCCT >701  83CGCCCGTCCCAGACCCTTA 30 60 66.8 −0.61 −5.02 CTCACTATGGA >703  84GCTACACGCCAGAGGCGCC 30 66.7 71 −7.27* −16.03* GCTACAGCGAT >706  85GAGATTGTACCCTACAGTC 30 46.7 60.4 −1.58 −4.26 CGATTACCGAT >715  86CGCAGTAAAAGGGCACAGG 30 43.3 60.1 −2.05 −19.3 TAATTACCTTA >718  87AGGGTGTCTTGAACTACTG 30 56.7 67.6 −3.43 −9.89 GCGCAGCCCAT >721  88CCGCAATCCGGTGACGGCC 30 76.7 74.8* −5.90* −16.50* GGACCGGCAGG >723  89TCGGCGGCGGGTAGTCAGT 30 66.7 70.3 −3.26 −6.97 TCGCTACCTGG >729  90CCAGGACTGCCGTGGCCCA 30 70 72.7* −2.93 −9.98 CGCACTCACGA >740  91TTGACGCAGGCCCCCGGGG 30 66.7 71.2 −2.92 −22.03* CGACTTCATAC >743  92CGAAAGGAGTTCGAGTGTA 30 56.7 64.7 −3.81 −12.90* TCCGGAAGGCG >745  93AGGCGCACTGCGACTTAGG 30 70 72.5 −4.68* −22.71* GCTAGCCCCCC >751  94GATGTGATCTGGACCCTAC 30 60 66.7 −3.51 −7.74 GGGAGGGGACA >754  95TGGGCTGGGGGAGTGAGTC 30 73.3 74.1* −10.51* −9.31 GCTCCCGCAGC >757  96AGTCCCAGATATGAGAGAA 30 43.3 60.4 −3.75 −4.39 GCGAAGCATAA >805  97CGTTTCAGCATCGATGTCC 25 40 55.7 −0.04 −13.62* TAAAAT >815  98ACTATTACACCACGTACCG 25 48 57.5 −2.43 −6.3 TAGGTC >816  99GGGCAACACCGCGAGCTAA 25 56 61.9 −0.21 −10.60* TTATCC >818 100GCGCGCGGCCGAGAATCGT 25 72 69.7 −1.77 −17.11* TGGAGG >826 101CGCGTCGGGCTTTCGTCTA 25 68 67 −1.3 −10.60* CCCTGG >829 102GGGCGGCCACCGGGGGACC 25 88 77.7 −3.41 −9.75 CTGCCC  U20   1GTCATATGCGACGATCTCA 20 50 52.8 0.38 −7.82 tag G (Std)

Step 4—Synthesis and In Vitro TMA Assay: Based on the screen for primerparameters, about 55-60 candidate sequences were identified as goodcandidate tag sequences (Table 2).

TABLE 2 Synthesized Universal non-T7 primer tags for  in vitro experimentation SEQ Tag Sequence ID Name # NO:Sequence (5′ to 3′) N1    6  4 GTCCCCATCGGAGGGCATCTTATCGTGCCT N2    8  5CCGCCCTCCTTCGCCCCCCGGTGAAATAAC N3   12  6 AATGCTCACCTCTATTCGGGACTTGAGTACN4   25  8 GTCGGAACGCCAGGTACAGTTAGCGCATCC N5   27 10GTGATGCTTTATGAGATTCCGGTCTCCGAC N6   28 11 GACGGTGCATCACCCGCATTTGCTGTAGCGN7   35 13 AAGCCAAAATTACAATCGATCCCTACCAAC N8   37 14ATCTTGCACCTTCCCAGATGTAAACCCCCT N9   82 21 CGGAGAATACCCTCGACTGTATCATATCGTN10   84 22 TTCATCGAGGTACATTGGTGCTATTCCATT N11   87 24AGGAGAACCAGCCTGGAGCGTTTAAGCATC N12   88 25GATGTCCTAAAATGAGGCGTGGCAATAGAG N13   89 26CAGAGTCATGTATACCCACTGTCGGTCGAA N14  104 27GTCAGGCTAGGGGGTTATCCCAGCAACGGC N15  116 29TTTTTGACAGTGATGAAGAGGGAGGTACGA N16  136 31CTATGGTTCGTTACTGAATCGAAAAGCCGC N17  138 32TAGCTATCAAAACAGGCGTCATCGGTTAAG N18  152 34CCTGCTTAGGGTCACTTAAACTACTGGCGC N19  155 35GGTGATGGCCCATACCGATCACGCCCGCAG N20  156 36CGGCAGGAGGGACTGCGATTTCCATAGAGC N21  159 37TGGCCGGAGAGAGGATAGGAAGCGGGACTA N22  163 39ACACATCCCAGGACTGCCGTGGCCTACGTA N23  174 42GACGAGCTTGTTCCAATTCCTCGAGCCGAG N24  179 43GTTGGGGAGGGGCACTACGACTTAGGGCTA N25  185 46GGCCGTCAATGTGTTTTGCACCCAACCGGA N26  193 48TCCCACGTCCTTCGACGCACACTGTAACTT N27 >301 49 TCATGTATCGCCCGTGGGTAAGCTCN28 >305 50 ATGTTATGGAGAGTGGGTTAGGCAA N29 >307 51ATGAGGGAGTAAGGAGATTAGGTTC N30 >313 53 GCCCAGCAGTTATACAATTCGTGGC N31 >31454 TTGGGCTCTCCAGTAGCCGAACAAA N32 >327 57 GTGGTTGCTACAGCCTAGCCTAGATN33 >339 59 AGGAGGAACCGGAAGATCTAATCTG N34 >343 61GCGACTGTGGCAACCCCATTTCGCA N35 >350 63 AGTTGGATGGATATCTCGCTCGTGA N36 >35664 CGCTGTCCTCTCTGACACTAAAGGT N37 >357 65 ATTTCAATAGTCAACCCGGTATCCAN38 >510 67 GGTTGGGGGGCTCGGCTCATGTATC N39 >517 69TAAGGAGACTAGGTTCCAATAGCTG N40 >523 70 TTACACAAATCGTGGGTTGGCCTCT N41 >53471 CGAAAGCGTTCCGCAGGACCCCCTT N42 >541 73 AAAGTCTGAGAATGAGTGATACCATN43 >544 74 ATATTGGTAGTTTTGTCCGCTGTAG N44 >549 75CGGAAGATCTAATCTGCACGCAATT N45 >608 76 GCGCCTCGTTGGGCAGAAGTTTGTGGAAATN46 >610 77 ATCTTCACCTACCGAGTTCTACGGGCCTAC N47 >613 78CCCACAACTTGCACCCGCTATGCGACCCTG N48 >629 82GATTAGTGGCCCAACGGGAACAAACTTCCT N49 >701 83CGCCCGTCCCAGACCCTTACTCACTATGGA N50 >706 85GAGATTGTACCCTACAGTCCGATTACCGAT N51 >715 86CGCAGTAAAAGGGCACAGGTAATTACCTTA N52 >718 87AGGGTGTCTTGAACTACTGGCGCAGCCCAT N53 >723 89TCGGCGGCGGGTAGTCAGTTCGCTACCTGG N54 >751 94GATGTGATCTGGACCCTACGGGAGGGGACA N55 >757 96AGTCCCAGATATGAGAGAAGCGAAGCATAA N56 >815 98 ACTATTACACCACGTACCGTAGGTC

The above sequences were synthesized either as the tag alone (as shownin Table 2 above) or as an oligonucleotide containing both atarget-specific sequence and a tag sequence (TS-tag) and were tested asdescribed in the following paragraphs:

Prostate cancer markers PCA3, PSA, T2:ERGa and CAP were used as targetnucleic acids in these examples. However, the target nucleic acids thatcan be used in the presently claimed methods are not hereby limited.

A TMA nucleic acid assay with PCA3 as a target nucleic acid was used toevaluate the candidate tag sequences. Testing was performed with 4replicates of 2 different PCA3 concentration levels. Results werecompared with those obtained using a tag which has been characterized inother amplification assays, the NT7 tag, also referred to as “U20.” Allassays used the same T7 provider.

Some representative real time curves from the NT7 tag evaluation areshown in FIGS. 4-6. Performance of the different tags varied in thisassay. Some performed approximately the same as U20, some performed muchmore poorly and some performed better. The best performing previouslyuncharacterized tags are N47, N48 and N49, which yielded dramaticdecreases in emergence times. These results demonstrate the power ofthis technique in identifying tags with preferable characteristics.

Example 2 Screen for T7 Tag Sequences

A similar strategy to Example 1 was performed for screening tags for usewith the T7 provider. A total of 22 sequences were identified forexperimental testing and shown in Table 3.

TABLE 3 Sequences Selected for Screening of T7 Tags SEQ Sequence Tag ID# Name NO: Sequence (5′ to 3′) >1003 T1 103 5′ TGGCTAATCCCG >1005 T2 1055′ CTGTGCTAGAGG >1006 T3 106 5′ CATGTACCAACG >1007 T4 1085′ TCGGTCGGACTA >1011 T5 109 5′ CCTCCCCCAAGC >1012 T6 1105′ GGGTTTGCTACG >1014 T7 111 5′ ATGTGCGCACAA >1022 T8 1125′ CGGGACTAGAGA >1026 T9 113 5′ AATCTCCGAGCG >1034 T10 1145′ AAGTGCAGGTTC >1042 T11 115 5′ TCCAGTTTAACC >1043 T12 1165′ TAGCCGCACAGG >1003b T13 104 5′ GCGTTGGCTAATCCCG >1006b T14 1075′ TCACCATGTACCAACG >1054 T15 117 5′ TATGAATGCGACCCGGAA >1063 T16 1185′ AACAATGGTCACTGCATC >1066 T17 119 5′ GGGCCGTTTCCCGGACATAA >1067 T18120 5′ AGGTTGAGTCCGCATCTGAA >1070 T19 1215′ TCGACCAAGAGCCGCTAGATGC >1076 T20 122 5′ AGCTCGTGTCAAGCCGTCGCCT >1083T21 123 5′ TGAAAGAGTTGTCAGTTTGCTGGT >1084 T22 1245′ TCAGGTAAAGGTTCCTCACGCTACC

The sequences of Table 3 were synthesized either as the tag alone (asshown in the table) plus a T7 promoter sequence[5′-aatttaatacgactcactatagggaga-3′] or as an oligonucleotide containingboth a target-specific sequence and a tag sequence plus the T7 promotersequence (i.e., construct of TS-tag=T7) and tested as described in thefollowing paragraphs:

The-TMA assay (which in this case used a directly hybridizedamplification oligomer complex with the “cPRO” configuration (USPublished Application 2008-0305482)) with PCA3 as a target was used inthe initial evaluation of the candidate tag sequences. That is,evaluated in a uniplex amplification reaction. Testing was performedwith 4 replicates of 2 different PCA3 target levels. Results werecompared with a previously characterized tag incorporated into a T7promoter-based amplification oligomer, referred to as “12 in[5′-CCACAACGGTTT-3′].” All assays in this initial evaluation used thesame NT7 primer comprising a previously characterized tag (U20).

Some representative real time curves from the T7 tag evaluation areshown in FIGS. 7-9. Performance of the different tags varied in thisassay. Some performed approximately the same as the standard 12 in, someperformed much more poorly and some performed better. The bestperforming previously uncharacterized tags in this system were T9, T14,T15, T16, T17, T20, T21 and T22, which yielded dramatic decreases inemergence times. One of the best performing tags—T15—was further testedusing PSA as a target nucleic acid, the results of which are shown inFIG. 10. Again, performance was dramatically improved using tagsequences identified by the current methods. These results demonstratethe power of this technique in identifying tags with preferablecharacteristics.

Example 3 Testing Combinations of NT7 and T7 tags

Combinations of NT7 and T7 tags are also tested using different TMAformats (see FIGS. 11-17). Combinations are identified whose performanceis superior to that of the standard U20/12 in pair, again demonstratingthe power of this technique in identifying tags with preferablecharacteristics.

Example 4 Further Refining Tag Sequences

As mentioned above, once good tag sequences have been identified,performance can be further optimized by the process of incrementalchanges and subsequent screening. An example of this is depicted inFIGS. 18-19, wherein the T7 tag T21 is systematically shortened and thentested in a TMA amplification assay. The T21 tag sequence was shortenedby removing residues from the 3′ end of the tag sequence. Shortening theT21 tag sequence by 2 nucleotides (“T21-2”) slightly improvedperformance. Shortening the T21 tag sequence by a total of 6 or 8nucleotides dramatically decreased its performance in this assay.Unexpectedly, shortening the T21 tag sequence even further by removing atotal of 10 or 12 nucleotides restored some (although not all) of theoriginal activity (especially 10 nucleotides shorter). This demonstratesthe ability of the method to identify sequences possessing unexpectedactivities, such as the example here wherein a very short tag yieldedgood performance. Further refining the tag sequence, as illustratedhere, is useful for “tuning” the tagged oligonucleotide to perform at acertain level. In some instances, a better performing taggedoligonucleotide is useful; while in other instances decreasing itsperformance is useful.

It should be noted that the differences in performance identified bythis method are useful in a wide variety of ways. For example, thedifferences in amplification kinetics identified are useful in balancingmultiple amplification reactions in a multiplex reaction in order thatno one (or more) reactions is so much faster than the others that it (orthey) unduly compete for essential shared components in the multiplexamplification mixture. Thus, the method of invention provides a meansfor evaluating and selecting useful tags by analyzing a variety ofperformance parameters, by which methods preferred sequencemodifications can be identified that would not have been a prioripredictable.

Example 5 Screen for NT7 Tag Sequences with Minimal Interference in thePCA3-PSA Duplex Template System Using the Reverse TMA with a Portion ofthe Amplification Oligomers Containing Tag Sequences

The example described below illustrates the use of the method to selectthe best tag sequences with minimal interference for use in a universalreverse TMA assay format using the PCA3-PSA target sequence combination.This example is intended only to demonstrate the use of this screeningmethod and does not limit the scope of application to only this assay.

Step 5—Screening in Uniplex: The 56 NT7 tags that were identified afterstep 3, and were then synthesized (Table 2) were screened foramplification of a PCA3 target nucleic acid in a uniplex format.

Each tag was screened in a uniplex RUh (reverse half universal TMA,where the universal sequence is on the primer member (NT7) of anamplification oligomer pair comprising a primer and a promoter-basedamplification oligomer) reaction for amplifying PCA3 target nucleicacid. Amplification results were evaluated qualitatively. Out of 56 NT7tags screened, 41 tag sequences optimally amplified PCA3 in this system(see FIG. 20 for representative examples). Separately, 38 NT7 tagsequences were designed and screened for amplification of a PSA targetnucleic acid. After qualitative evaluation, 16 of these tagssufficiently amplified PSA (see FIG. 21 for representative examples).

Step 6—Screening Using Duplex Amplification Oligos: Each of the 41 PCA3tags passing qualitative criteria in uniplex were then screened induplex oligo format. PCA3 amplification driven by each tag was observedin presence of PSA oligos containing the U20 standard tag sequence.Similarly, PSA amplification driven by the previously characterized U20tag was evaluated in the presence of PCA3 amplification oligos with eachof 41 tags (see FIGS. 22 and 23 for representative examples). Theseduplex oligo screens were evaluated qualitatively, and 12 PCA3 tags wereconfirmed to work well in the presence of PSA oligos comprising standardtag U20.

Each of 16 tag sequences that passed qualitative criteria for PSAamplification in uniplex were then screened against several good tagsfor PCA3. 27 of these unique tag combinations successfully amplifiedPCA3 and PSA under duplex oligos conditions.

Step 7—Screening Using Duplex Amplification Oligos and Target NucleicAcids: All tag combinations passing qualitative criteria under duplexoligos conditions were subsequently screened in duplex oligos and targetsequences format. Amplification of 10³ copies of PCA3 was evaluated inthe presence of 10⁶ copies of PSA, and 10³ copies PSA was evaluated inthe presence of 10⁶ copies of PCA3. This target input window was chosenbased on the interference observed in the standard tag sequence (U20)universal system; specifically, 1000 copies of either PSA or PCA3 targetnucleic acid amplified only weakly in the presence of 3-log greatercopies of the other target nucleic acid when using a U20 tag (FIG. 24).The levels of template interference with each new tag combination werequalitatively determined relative to the amplification with the U20standard tag.

Step 8—Interference Analysis: The interference analysis method was thenused to quantitate the magnitude of target sequence interference(I-values) for each tag combination screened in duplex oligos+templatescondition. See FIGS. 25 and 26. The tested combinations are shown inFIG. 27 together with the results of the screening test. The lowestI-value of 19.97 for an Analyte Interference Score was seen for the tagsequence combination, Tag N54 (PCA3) and standard tag U20 (PSA). The top10 tag combinations displaying minimal I-value, are listed in Table 5. Asummary of results from all the screens performed for this example isgiven in FIG. 27.

TABLE 5 displays the top 10 tags demonstrating minimal multiplexinterference relative to the standard tag U20. PCA3 Tag PSA Tag DeltaPCA3 Delta PSA Score U20 U20 27 24 51 N54 U20 12  7 19 N54 N209 16  4 20N34 N201  9 12 21 N14 U20  8 13 21 N54 N226 19  3 22 N54 N216 15  8 23N14 N216 12 12 24 N34 N216  6 20 25 N14 N207  6 22 28 N14 N201  9 24 33

The best tag combination obtained from this screen was N54/U20. Finally,using the combinations of tags that were selected from the screens, tagN54 (PCA3) and tag U20 (PSA), it was demonstrated that the interferenceobserved was minimal in a Triplex system (PCA3/PSA/IC) compared to thestandard system (FIGS. 28 to 31). Reactions conditions in this triplexsystem were generally set up as follows. For a first set of triplexreactions the non-T7 amplification oligomers for each target nucleicacid (PCA3, PSA and Internal Control) were tagged with the U20 tagsequence (see FIG. 28). Each target capture reaction contained 5 pmol ofeach target capture oligomer, 5 pmol of each blocker and varied copynumbers of the target nucleic acids according to the chart in FIG. 28along with internal control. Target capture reactions were performedresulting in capture of the target nucleic acid hybridized with a non-T7amplification oligomer and a blocker. The captured nucleic acids werethen placed into an amplification reaction containing 2.8 pmol, 1.3 pmoland 0.6 pmol of PCA3 T7 amp oligos, PSA T7 amp oligos and internalcontrol T7 amp oligos, respectively; 20 pmol each of PCA3 torch and PSAtorch; 10 pmol of internal control torch; and 10 pmol of a non-T7amplification oligomer targeting the complement of the U20 tag sequence.A real time TMA reaction was performed and the results are shown in FIG.29. Two nearly identical assays were also performed, the differencesbeing that the non-T7 amplification oligomer targeting PCA3 was taggedwith N54 rather than U20. Each of these additional assays contained thesame oligomers and target nucleic acids. However, to off-set the impactof a more robust amplification of one of the targets within themultiplex, the T7 concentrations were adjusted. In these additional tworeactions, the PCA3 reaction included 3.0-3.8 pmol T7 and 15-20 pmol oftorch; the PSA reaction included 15-20 pmol torch and the internalcontrol included 15-20 pmol torch. Other differences compared to thefull U20 reaction included using 7.5 or 15 pmol of non-T7 targeting thecomplement of U20 or N54, respectively. Target copy numbers for thesethree reactions are shown in FIGS. 28 and 30. Results are shown in FIGS.29 and 31.

The system using the N54 tag was able to amplify 102 copies of PCA3 and2491 copies PSA in presence of the highest calibrator level of thecompeting analyte, whereas the U20 universal “standard” system would notunder a balanced set of condition (see FIG. 29). An ‘unbalanced’ systemwas tested with several different low copy levels in presence of highcopy levels of analyte, and the N54/U20/U20 system demonstrated verylittle interference compared to the U20 standard triplex system (seeFIG. 31). Therefore one the advantage of using the tag identificationsystem disclosed herein is that the unbalanced system achieved the samelevel of quantitation across the dynamic range without interference asdid a balanced system.

Thus, the screening method described herein successfully identified tagsequences with minimal multiplex interference in a model system. Ingeneral, improved sequence tags provide selective advantage to anyunder-performing amplification system, and can be selected for a varietyof desired properties like speed of amplification, performance, etc,along with minimal interference. In addition, screens can be modifiedand adapted for identifying improved tags, for alternate TMA formats,other isothermal and non-isothermal amplification formats, includingPCR, and in higher plex amplifications. The methods described herein fortag selection can be readily automated to provide a high throughputcombinatorial method that quickly screens for tag sequences for severaltypes of assays.

Example 7 Identification and Selection of Tags that are Used inConjunction with T2:ERGa Target Sequence in T2:ERGa/PSA/IC Triplex TMAAssay System

T2:ERGa is a particularly challenging target nucleic acid foramplification using TMA due, in part, to its Guanine-rich sequence. Thestandard tag (U20) interferes with T2:ERGa amplification and is not ableto amplify and detect the analyte in a universal TMA format. The tagsdescribed herein which were identified using the disclosed inventionmethod are able to detect and quantitate T2:ERGa and PSA with sufficientsensitivity, accuracy and precision in the reverse TMA reaction whereina portion of the amplification oligomers used contain tag sequences.

Previously known tags were not able to amplify T2:ERGa with desiredsensitivity and precision. A comparison of the assay performance withthe standard tag (U20) and tag N42 is shown in FIG. 32.

The N42 tag, for example, was capable of amplifying T2:ERGa in uniplexas well as in a triplex assay that comprised PSA target sequence and aninternal control sequence along with T2:ERGa target nucleic acid. Thetriplex assay performed by using the N42 tag to amplify all the threetarget sequences in a one tube multiplex format yielded the desiredperformance characteristics (see FIGS. 33 and 34).

The N42 tag was also found to be compatible with several other uniquetag sequence combinations which are useful in conjunction with T2:ERGa.A couple of examples of assays performed with compatible unique tagsequences in the T2:ERGa/PSA/IC system are shown in FIGS. 35 to 38.

All the tag sequences and combinations disclosed in FIGS. 32-38 areuseful for amplification and quantitative detection of T2:ERGa and PSAtemplates in the T2:ERGa/PSA/IC triplex assay in pure system (using IVT)as well as in clinical urine samples.

TABLE 4 Summary and cross-identification of the sequencesdescribed herein and in the Sequence Listing. Oligo SEQ ID Name NO:Sequence 5′→3′ Function U20 tag   1 GTCATATGCGACGATCTCAG tag (Std) 1   2CCCCGTCAAACAAAAACGGGAGCGTGTACC tag 4   3 CCATAGGCCTTCTGCACTGCTCCATATACCtag 6/N1   4 GTCCCCATCGGAGGGCATCTTATCGTGCCT tag 8/N2   5CCGCCCTCCTTCGCCCCCCGGTGAAATAAC tag 12/N3   6AATGCTCACCTCTATTCGGGACTTGAGTAC tag 21   7 CCCGCGCACCACCTCCATCACGCAGAAGAGtag 25/N4   8 GTCGGAACGCCAGGTACAGTTAGCGCATCC tag 26   9AAGTCACTGGCCAGCATAATGCGTGAAGGG tag 27/N5  10GTGATGCTTTATGAGATTCCGGTCTCCGAC tag 28/N6  11GACGGTGCATCACCCGCATTTGCTGTAGCG tag 34  12 AGAATTCTTGCAGGTAGAGGTCCCCTCATTtag 35/N7  13 AAGCCAAAATTACAATCGATCCCTACCAAC tag 37/N8  14ATCTTGCACCTTCCCAGATGTAAACCCCCT tag 42  15 GAAGCGGCAGCTCAGCCGGTTCTCGGAGAGtag 43  16 GCACGCGGGCTCCTTGGGACACTATGATTG tag 61  17CCCATCAGGACAGTCAGCTGCCCACGAATT tag 78  18 CTTTAGTGCGGTAGGACCGAGACTACCGTGtag 79  19 TTATGTGCCAGCTGGGCCTAAGGCTCCGGG tag 80  20GACTCTCCTAGGGCGTTCGTCTGGGACTGC tag 82/N9  21CGGAGAATACCCTCGACTGTATCATATCGT tag 84/N10  22TTCATCGAGGTACATTGGTGCTATTCCATT tag 86  23 TACCACCTGGTTCAAGGTGTGCCGTACGCGtag 87/N11  24 AGGAGAACCAGCCTGGAGCGTTTAAGCATC tag 88/N12  25GATGTCCTAAAATGAGGCGTGGCAATAGAG tag 89/N13  26CAGAGTCATGTATACCCACTGTCGGTCGAA tag 104/N14  27GTCAGGCTAGGGGGTTATCCCAGCAACGGC tag 106  28TGGGTTCTGCTAACCGGTGCCGTTCTTAAC tag 116/N15  29TTTTTGACAGTGATGAAGAGGGAGGTACGA tag 133  30GAGAACTCGCGCTCCCTCACTCCGTTTAGA tag 136/N16  31CTATGGTTCGTTACTGAATCGAAAAGCCGC tag 138/N17  32TAGCTATCAAAACAGGCGTCATCGGTTAAG tag 145  33AGGACGCTGACACCGTTGGGGTAAAGCGTG tag 152/N18  34CCTGCTTAGGGTCACTTAAACTACTGGCGC tag 155/N19  35GGTGATGGCCCATACCGATCACGCCCGCAG tag 156/N20  36CGGCAGGAGGGACTGCGATTTCCATAGAGC tag 159/N21  37TGGCCGGAGAGAGGATAGGAAGCGGGACTA tag 161  38TAGCAGGTGTCTCGGTCCTCAACTGCAAAC tag 163/N22  39ACACATCCCAGGACTGCCGTGGCCTACGTA tag 171  40GTGCTAGCCCGGGCCCTTCTTAACTCGGGA tag 172  41CGGAATCTGAACATCTATCAGAGCCGCGCT tag 174/N23  42GACGAGCTTGTTCCAATTCCTCGAGCCGAG tag 179/N24  43GTTGGGGAGGGGCACTACGACTTAGGGCTA tag 182  44AATGTGGACGGCCGCTCCGTACTTCTGACA tag 183  45AGGGCCAGCAGCTGGTTCCTTCGCCAGTTA tag 185/N25  46GGCCGTCAATGTGTTTTGCACCCAACCGGA tag 188  47CAGTGACTGGGCTAGTGAAGTGAGTCACAG tag 193/N26  48TCCCACGTCCTTCGACGCACACTGTAACTT tag 301/N27  49 TCATGTATCGCCCGTGGGTAAGCTCtag 305/N28  50 ATGTTATGGAGAGTGGGTTAGGCAA tag 307/N29  51ATGAGGGAGTAAGGAGATTAGGTTC tag 309  52 CATGCTGCCCGCATACACTTGCGGG tag313/N30  53 GCCCAGCAGTTATACAATTCGTGGC tag 314/N31  54TTGGGCTCTCCAGTAGCCGAACAAA tag 316  55 TGACGTTAAACGCAATCCGCGTAAA tag 325 56 GTCGCCATTCAGGACACGCGAAACT tag 327/N32  57 GTGGTTGCTACAGCCTAGCCTAGATtag 336  58 CCACTTTTCATTCCGAGTCCACGCG tag 339/N33  59AGGAGGAACCGGAAGATCTAATCTG tag 342  60 CCAATGCTTTCAAATAACCCGTTCT tag343/N34  61 GCGACTGTGGCAACCCCATTTCGCA tag 346  62AAAAAACGGAGGAGTCGAACCTTGG tag 350/N35  63 AGTTGGATGGATATCTCGCTCGTGA tag356/N36  64 CGCTGTCCTCTCTGACACTAAAGGT tag 357/N37  65ATTTCAATAGTCAACCCGGTATCCA tag 505  66 TTCGCGCCAGCGACCCCACTTATGA tag510/N38  67 GGTTGGGGGGCTCGGCTCATGTATC tag 516  68ATGATGCTGAATCGCGATGGGGGGG tag 517/N39  69 TAAGGAGACTAGGTTCCAATAGCTG tag523/N40  70 TTACACAAATCGTGGGTTGGCCTCT tag 534/N41  71CGAAAGCGTTCCGCAGGACCCCCTT tag 538  72 CACCCTTGGACACGTGGAAGTGGGC tag541/N42  73 AAAGTCTGAGAATGAGTGATACCAT tag 544/N43  74ATATTGGTAGTTTTGTCCGCTGTAG tag 549/N44  75 CGGAAGATCTAATCTGCACGCAATT tag608/N45  76 GCGCCTCGTTGGGCAGAAGTTTGTGGAAAT tag 610/N46  77ATCTTCACCTACCGAGTTCTACGGGCCTAC tag 613/N47  78CCCACAACTTGCACCCGCTATGCGACCCTG tag 618  79GCCCAGGAGCTCTCCTGGGTAACAGTAGCG tag 620  80CACGGCCCCCAGGCGGCGTATCAGGGATGA tag 623  81TCCCCGGCACGGACCGCAAGGGACCAAAGC tag 629/N48  82GATTAGTGGCCCAACGGGAACAAACTTCCT tag 701/N49  83CGCCCGTCCCAGACCCTTACTCACTATGGA tag 703  84GCTACACGCCAGAGGCGCCGCTACAGCGAT tag 706/N50  85GAGATTGTACCCTACAGTCCGATTACCGAT tag 715/N51  86CGCAGTAAAAGGGCACAGGTAATTACCTTA tag 718/N52  87AGGGTGTCTTGAACTACTGGCGCAGCCCAT tag 721  88CCGCAATCCGGTGACGGCCGGACCGGCAGG tag 723/N53  89TCGGCGGCGGGTAGTCAGTTCGCTACCTGG tag 729  90CCAGGACTGCCGTGGCCCACGCACTCACGA tag 740  91TTGACGCAGGCCCCCGGGGCGACTTCATAC tag 743  92CGAAAGGAGTTCGAGTGTATCCGGAAGGCG tag 745  93AGGCGCACTGCGACTTAGGGCTAGCCCCCC tag 751/N54  94GATGTGATCTGGACCCTACGGGAGGGGACA tag 754  95TGGGCTGGGGGAGTGAGTCGCTCCCGCAGC tag 757/N55  96AGTCCCAGATATGAGAGAAGCGAAGCATAA tag 805  97 CGTTTCAGCATCGATGTCCTAAAAT tag815/N56  98 ACTATTACACCACGTACCGTAGGTC tag 816  99GGGCAACACCGCGAGCTAATTATCC tag 818 100 GCGCGCGGCCGAGAATCGTTGGAGG tag 826101 CGCGTCGGGCTTTCGTCTACCCTGG tag 829 102 GGGCGGCCACCGGGGGACCCTGCCC tag1003/T1 103 TGGCTAATCCCG tag 1003b/T13 104 GCGTTGGCTAATCCCG tag 1005/T2105 CTGTGCTAGAGG tag 1006/T3 106 CATGTACCAACG tag 1006b/T14 107TCACCATGTACCAACG tag 1007/T4 108 TCGGTCGGACTA tag 1011/T5 109CCTCCCCCAAGC tag 1012/T6 110 GGGTTTGCTACG tag 1014/T7 111 ATGTGCGCACAAtag 1022/T8 112 CGGGACTAGAGA tag 1026/T9 113 AATCTCCGAGCG tag 1034/T10114 AAGTGCAGGTTC tag 1042/T11 115 TCCAGTTTAACC tag 1043/T12 116TAGCCGCACAGG tag 1054/T15 117 TATGAATGCGACCCGGAA tag 1063/T16 118AACAATGGTCACTGCATC tag 1066/T17 119 GGGCCGTTTCCCGGACATAA tag 1067/T18120 AGGTTGAGTCCGCATCTGAA tag 1070/T19 121 TCGACCAAGAGCCGCTAGATGC tag1076/T20 122 AGCTCGTGTCAAGCCGTCGCCT tag 1083/T21 123TGAAAGAGTTGTCAGTTTGCTGGT tag 1084/T22 124 TCAGGTAAAGGTTCCTCACGCTACC tagN200 125 CCCATAACTTGGTGCGAATACGGGT tag N201 126CGTAGCAATGTTCGTCTGACTATGA tag N202 127 CAACTACGGGGATTCTTGGAGAGCC tagN203 128 GTGTAGTATTAGCAAACGATAAGTC tag N204 129CGGGGGCTGGGAATCTGTGACATGA tag N205 130 TGCCTGTCGATCCATAGGACTCGTG tagN206 131 GAAATGTCCGGGGCCAAAGACAACC tag N207 132CTGACATAGTATAGCATAGATATTG tag N208 133 GAATTTATAGATACTGCCAATCTAG tagN209 134 ATCAGTTGGACAGAGGGCTGTGTTA tag N210 135CTTCTAGAGAAGAAGAGTACTGACT tag N211 136 GGTTCAGTTGTAACCATATACTTAC tagN212 137 AATGACGTAGCTATGTATTTTGCAC tag N213 138AGGTAGCCAACGGGTTTCACATTTC tag N214 139 GCGTAAACTACGATGGCACCTACTC tagN215 140 CTCATAACTTGGTGCGAATACGGGT tag N216 141TGTAGCAATGTTCGTCTGACTATGA tag N217 142 TAAAATAGTACAGCTACTGGTGATC tagN218 143 CAACTACGAGGATTTTTAGAGAGCC tag N219 144ATGTAGTATTAGCAAACAATAAGTC tag N220 145 AATTGAATGGAGTCTGATCAATCTT tagN221 146 GAAGTTGGAGGATTAACGTGGGAAT tag N222 147GGTTTACTATTGTCTCTAATGGGAG tag N223 148 TTGACATAGTATAGCATAGATATTG tagN224 149 CAGATAACTTACCTACATTGAAAGT tag N225 150TATAGACGACTATTCCGACTAGCAA tag N226 151 GAATTTATAGATACTACCAATCTAG tagN227 152 ATTAATTGGACAGAGGGCTGTGTTA tag N228 153CTGTTGCCACTCTTTAGAAAGATTA tag N301 154 ACTACAATAATACCAACTATTTGCC tagN302 155 GATACTAAATAACAACTTAGTTTTT tag N303 156TAGATTTCATTCCGAGTCCACATGT tag N304 157 AACTCTAATATAAGATATCAAGTTA tagN305 158 ATTGTTAAAGTAGACTAATTATCTA tag N306 159GAAGGAACTGGAAGATTTAATTTGC tag N307 160 ACGCAATTAATATACATATTTATAC tagN308 161 CAATTATGCGAATTCCATTTCACAT tag N309 162CTTATGAGATGTTAGATATAGTATT tag N310 163 CTTTTACAATATCAGACTTTAGCAA tagN311 164 GATGTAGACGGATTCCATAGAATTT tag N312 165AATGATTGTGTGGAGTACAAACCAA tag N313 166 TTTTTTTGGCGTAAAGTCTAGAGTT tagN314 167 ATCACGTAAGACCACTGTTAGTATA tag N315 168CTTTAATAGTCAAACCGATATCCAT tag N316 169 CAGTCAAGTGATGGACTCTAACACA tagN317 170 TCATTAGCGGAAAAAACTGACCTTC tag N318 171CTCCTATCCTTCGCCACAACTTTAG tag N319 172 TTGCTTTGAGATTGAAATATAAAAG tagN320 173 ATCATATACAGTGCCAGGGAACAAC tag N321 174ACTTGTAGAAATACCTTATAAAGTT tag N322 175 ATTCTTGATGTATGTAGAGTCCTAA tagN323 176 TGATATCGAATACATAAGTACTCGA tag N324 177ATGACTGAATTGCTTACACATTTAA tag N325 178 AAAAACAATTAGTATATAACTATTA tagN326 179 TAATAGTGTCATCGGCTCCACTTAT tag N327 180TACAATCAAAACGTGAAGTTATTGA tag N328 181 GTTTAGTTATTGACTTGTAGATAGT tagN329 182 CTCGACACCGAGTGCTAGATCAACG tag N330 183ACCCGGACATATTGGCTATTCAAAC tag N331 184 AATATTTAAAAGCCTGGTTTATGTA tagN332 185 CTTTAGTGCCGATTTACGGCCTTGG tag N333 186GGTAAGATAACGAAGTTTTAATAGC tag N334 187 TGCTTTGACACTGTTCATTATACCG tagN335 188 TTTTCTTTTACCCACTGGTGAAATA tag N336 189TCAAGATTGTCCTTGATTGTTGAAT tag N337 190 AAAGATCTGATTAACTTATAACAGA tagN338 191 ATGAATAAATCTTGTAAAGTGTGGC tag N339 192AGCTACACTAAACCTAGAATGATCT tag N340 193 CTTCAATTTGAGACTTGAAATCTAA tagN341 194 GTTTCACTCAGTGTAGACATCATCC tag N342 195TGGTATCTGAATTACTGCTTTGTCA tag N343 196 AAGTGTCTATTATCCTTAAACGCAT tagN344 197 ATCTCGCATAATAACTCCTCAATAT tag N345 198GAGTTAGTCTTGTGCTCACGGAATT tag N346 199 AAATGTTAGTTAGCTCGTTCAAGTA tagN347 200 AAAGTTCTTCACACTACGTCAAAAT tag N348 201AAAAAAATGGTTGTAACAAAAAAAA tag 202 GGCTCATCGATGACCCAAGATGGCGGC Promoter203 TCTCCCTATAGTGAGTCGTATTAAATT Reverse Complement Promoter 204GTCTAAGTAGTGACATGTTT PCA3 target specific portion for promoter primer205 TGGCTAATCCCGGTCTAAGTAGTGACATGTTT T1/PCA3 T1 206GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCG Prom/T1 T1b 207GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCGGT Prom/T1/ CTAAGTAGTGACATGTTTPCA3 208 CTGTGCTAGAGGGTCTAAGTAGTGACATGTTT T2/PCA3 T2 209GGCTCATCGATGACCCAAGATGGCGGCCTGTGCTAGAGG Prom/T2 T2b 210GGCTCATCGATGACCCAAGATGGCGGCCTGTGCTAGAGGGT Prom/T2/ CTAAGTAGTGACATGTTTPCA3 211 CATGTACCAACGGTCTAAGTAGTGACATGTTT T3/PCA3 T3 212GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACG Prom/T3 T3b 213GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACGGT Prom/T3/ CTAAGTAGTGACATGTTTPCA3 214 TCGGTCGGACTAGTCTAAGTAGTGACATGTTT T4/PCA3 T4 215GGCTCATCGATGACCCAAGATGGCGGCTCGGTCGGACTA Prom/T4 T4b 216GGCTCATCGATGACCCAAGATGGCGGCTCGGTCGGACTAGT Prom/T4/ CTAAGTAGTGACATGTTTPCA3 217 CCTCCCCCAAGCGTCTAAGTAGTGACATGTTT T5/PCA3 T5 218GGCTCATCGATGACCCAAGATGGCGGCCCTCCCCCAAGC Prom/T5 T5b 219GGCTCATCGATGACCCAAGATGGCGGCCCTCCCCCAAGCGT Prom/T5/ CTAAGTAGTGACATGTTTPCA3 220 GGGTTTGCTACGGTCTAAGTAGTGACATGTTT T6/PCA3 T6 221GGCTCATCGATGACCCAAGATGGCGGCGGGTTTGCTACG Prom/T6 T6b 222GGCTCATCGATGACCCAAGATGGCGGCGGGTTTGCTACGGT Prom/T6/ CTAAGTAGTGACATGTTTPCA3 223 ATGTGCGCACAAGTCTAAGTAGTGACATGTTT T7/PCA3 T7 224GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAA Prom/T7 T7b 225GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAAGT Prom/T7/ CTAAGTAGTGACATGTTTPCA3 226 CGGGACTAGAGAGTCTAAGTAGTGACATGTTT T8/PCA3 T8 227GGCTCATCGATGACCCAAGATGGCGGCCGGGACTAGAGA Prom/T8 T8b 228GGCTCATCGATGACCCAAGATGGCGGCCGGGACTAGAGAGT Prom/T8/ CTAAGTAGTGACATGTTTPCA3 229 AATCTCCGAGCGGTCTAAGTAGTGACATGTTT T9/PCA3 T9 230GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCG Prom/T9 T9b 231GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCGGT Prom/T9/ CTAAGTAGTGACATGTTTPCA3 232 AAGTGCAGGTTCGTCTAAGTAGTGACATGTTT T10/PCA3 T10 233GGCTCATCGATGACCCAAGATGGCGGCAAGTGCAGGTTC Prom/T10 T10b 234GGCTCATCGATGACCCAAGATGGCGGCAAGTGCAGGTTCGT Prom/T10/ CTAAGTAGTGACATGTTTPCA3 235 TCCAGTTTAACCGTCTAAGTAGTGACATGTTT T11/PCA3 T11 236GGCTCATCGATGACCCAAGATGGCGGCTCCAGTTTAACC Prom/T11 T11b 237GGCTCATCGATGACCCAAGATGGCGGCTCCAGTTTAACCGT Prom/T11/ CTAAGTAGTGACATGTTTPCA3 238 TAGCCGCACAGGGTCTAAGTAGTGACATGTTT T12/PCA3 T12 239GGCTCATCGATGACCCAAGATGGCGGCTAGCCGCACAGG Prom/T12 T12b 240GGCTCATCGATGACCCAAGATGGCGGCTAGCCGCACAGGGT Prom/T12/ CTAAGTAGTGACATGTTTPCA3 241 GCGTTGGCTAATCCCGGTCTAAGTAGTGACATGTTT T13/PCA3 T13 242GGCTCATCGATGACCCAAGATGGCGGCGCGTTGGCTAATCC Prom/T13 CG T13b 243GGCTCATCGATGACCCAAGATGGCGGCGCGTTGGCTAATCC Prom/T13/CGGTCTAAGTAGTGACATGTTT PCA3 244 TCACCATGTACCAACGGTCTAAGTAGTGACATGTTTT14/PCA3 T14 245 GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14 CGT14b 246 GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14/CGGTCTAAGTAGTGACATGTTT PCA3 247 TATGAATGCGACCCGGAAGTCTAAGTAGTGACATGTTTT15/PCA3 T15 248 GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15 GGAAT15b 249 GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15/GGAAGTCTAAGTAGTGACATGTTT PCA3 250 AACAATGGTCACTGCATCGTCTAAGTAGTGACATGTTTT16/PCA3 T16 251 GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16 CATCT16b 252 GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16/CATCGTCTAAGTAGTGACATGTTT PCA3 253GGGCCGTTTCCCGGACATAAGTCTAAGTAGTGACATGTTT T17/PCA3 T17 254GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17 ACATAA T17b 255GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17/ACATAAGTCTAAGTAGTGACATGTTT PCA3 256AGGTTGAGTCCGCATCTGAAGTCTAAGTAGTGACATGTTT T18/PCA3 T18 257GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18 TCTGAA T18b 258GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18/TCTGAAGTCTAAGTAGTGACATGTTT PCA3 259TCGACCAAGAGCCGCTAGATGCGTCTAAGTAGTGACATGTT T19/PCA3 T T19 260GGCTCATCGATGACCCAAGATGGCGGCTCGACCAAGAGCCG Prom/T19 CTAGATGC T19b 261GGCTCATCGATGACCCAAGATGGCGGCTCGACCAAGAGCCG Prom/T19/CTAGATGCGTCTAAGTAGTGACATGTTT PCA3 262AGCTCGTGTCAAGCCGTCGCCTGTCTAAGTAGTGACATGTT T20/PCA3 T T20 263GGCTCATCGATGACCCAAGATGGCGGCAGCTCGTGTCAAGC Prom/T20 CGTCGCCT T20b 264GGCTCATCGATGACCCAAGATGGCGGCAGCTCGTGTCAAGC Prom/T20/CGTCGCCTGTCTAAGTAGTGACATGTTT PCA3 265TGAAAGAGTTGTCAGTTTGCTGGTGTCTAAGTAGTGACATG T21/PCA3 TTT T21 266GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21 GTTTGCTGGT T21b 267GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21/GTTTGCTGGTGTCTAAGTAGTGACATGTTT PCA3 268TCAGGTAAAGGTTCCTCACGCTACCGTCTAAGTAGTGACAT T22/PCA3 GTTT T22 269GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22 CTCACGCTACC T22b 270GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22/CTCACGCTACCGTCTAAGTAGTGACATGTTT PCA3 271 GGCTCATCGATGACCCAAGATGGCGGCPCA3 target specific sequence for non- promoter primer RPCA321 272GTCATATGCGACGATCTCAGGGCTCATCGATGACCCAAGAT U20/PCA3 U20 GGCGGC N1b 273GTCCCCATCGGAGGGCATCTTATCGTGCCTGGCTCATCGAT N1/PCA3 GACCCAAGATGGCGGCnon-prom N2b 274 CCGCCCTCCTTCGCCCCCCGGTGAAATAACGGCTCATCGAT N2/PCA3GACCCAAGATGGCGGC non-prom N3b 275AATGCTCACCTCTATTCGGGACTTGAGTACGGCTCATCGAT N3/PCA3 GACCCAAGATGGCGGCnon-prom N4b 276 GTCGGAACGCCAGGTACAGTTAGCGCATCCGGCTCATCGAT N4/PCA3GACCCAAGATGGCGGC non-prom N5b 277GTGATGCTTTATGAGATTCCGGTCTCCGACGGCTCATCGAT N5/PCA3 GACCCAAGATGGCGGCnon-prom N6b 278 GACGGTGCATCACCCGCATTTGCTGTAGCGGGCTCATCGAT  N6/PCA3GACCCAAGATGGCGGC non-prom N7b 279AAGCCAAAATTACAATCGATCCCTACCAACGGCTCATCGAT N7/PCA3 GACCCAAGATGGCGGCnon-prom N8b 280 ATCTTGCACCTTCCCAGATGTAAACCCCCTGGCTCATCGAT N8/PCA3GACCCAAGATGGCGGC non-prom N9b 281CGGAGAATACCCTCGACTGTATCATATCGTGGCTCATCGAT N9/PCA3 GACCCAAGATGGCGGCnon-prom N10b 282 TTCATCGAGGTACATTGGTGCTATTCCATTGGCTCATCGAT N10/PCA3GACCCAAGATGGCGGC non-prom N11b 283AGGAGAACCAGCCTGGAGCGTTTAAGCATCGGCTCATCGAT N11/PCA3 GACCCAAGATGGCGGCnon-prom N12b 284 GATGTCCTAAAATGAGGCGTGGCAATAGAGGGCTCATCGAT N12/PCA3GACCCAAGATGGCGGC non-prom N13b 285CAGAGTCATGTATACCCACTGTCGGTCGAAGGCTCATCGAT N13/PCA3 GACCCAAGATGGCGGCnon-prom N14b 286 GTCAGGCTAGGGGGTTATCCCAGCAACGGCGGCTCATCGAT N14/PCA3GACCCAAGATGGCGGC non-prom N15b 287TTTTTGACAGTGATGAAGAGGGAGGTACGAGGCTCATCGAT N15/PCA3 GACCCAAGATGGCGGCnon-prom N16b 288 CTATGGTTCGTTACTGAATCGAAAAGCCGCGGCTCATCGAT N16/PCA3GACCCAAGATGGCGGC non-prom N17b 289TAGCTATCAAAACAGGCGTCATCGGTTAAGGGCTCATCGAT N17/PCA3 GACCCAAGATGGCGGCnon-prom N18b 290 CCTGCTTAGGGTCACTTAAACTACTGGCGCGGCTCATCGAT N18/PCA3GACCCAAGATGGCGGC non-prom N19b 291GGTGATGGCCCATACCGATCACGCCCGCAGGGCTCATCGAT N19/PCA3 GACCCAAGATGGCGGCnon-prom N20b 292 CGGCAGGAGGGACTGCGATTTCCATAGAGCGGCTCATCGAT N20/PCA3GACCCAAGATGGCGGC non-prom N21b 293TGGCCGGAGAGAGGATAGGAAGCGGGACTAGGCTCATCGAT N21/PCA3 GACCCAAGATGGCGGCnon-prom N22b 294 ACACATCCCAGGACTGCCGTGGCCTACGTAGGCTCATCGAT N22/PCA3GACCCAAGATGGCGGC non-prom N23b 295GACGAGCTTGTTCCAATTCCTCGAGCCGAGGGCTCATCGAT N23/PCA3 GACCCAAGATGGCGGCnon-prom N24b 296 GTTGGGGAGGGGCACTACGACTTAGGGCTAGGCTCATCGAT N24/PCA3GACCCAAGATGGCGGC non-prom N25b 297GGCCGTCAATGTGTTTTGCACCCAACCGGAGGCTCATCGAT N25/PCA3 GACCCAAGATGGCGGCnon-prom N26b 298 TCCCACGTCCTTCGACGCACACTGTAACTTGGCTCATCGAT N26/PCA3GACCCAAGATGGCGGC non-prom N27b 299TCATGTATCGCCCGTGGGTAAGCTCGGCTCATCGATGACCC N27/PCA3 AAGATGGCGGC non-promN28b 300 ATGTTATGGAGAGTGGGTTAGGCAAGGCTCATCGATGACCC N28/PCA3 AAGATGGCGGCnon-prom N29b 301 ATGAGGGAGTAAGGAGATTAGGTTCGGCTCATCGATGACCC N29/PCA3AAGATGGCGGC non-prom N30b 302 GCCCAGCAGTTATACAATTCGTGGCGGCTCATCGATGACCCN30/PCA3 AAGATGGCGGC non-prom N31b 303TTGGGCTCTCCAGTAGCCGAACAAAGGCTCATCGATGACCC N31/PCA3 AAGATGGCGGC non-promN32b 304 GTGGTTGCTACAGCCTAGCCTAGATGGCTCATCGATGACCC N32/PCA3 AAGATGGCGGCnon-prom N33b 305 AGGAGGAACCGGAAGATCTAATCTGGGCTCATCGATGACCC N33/PCA3AAGATGGCGGC non-prom N34b 306 GCGACTGTGGCAACCCCATTTCGCAGGCTCATCGATGACCCN34/PCA3 AAGATGGCGGC non-prom N35b 307AGTTGGATGGATATCTCGCTCGTGAGGCTCATCGATGACCC N35/PCA3 AAGATGGCGGC non-promN36b 308 CGCTGTCCTCTCTGACACTAAAGGTGGCTCATCGATGACCC N36/PCA3 AAGATGGCGGCnon-prom N37b 309 ATTTCAATAGTCAACCCGGTATCCAGGCTCATCGATGACCC N37/PCA3AAGATGGCGGC non-prom N38b 310 GGTTGGGGGGCTCGGCTCATGTATCGGCTCATCGATGACCCN38/PCA3 AAGATGGCGGC non-prom N39b 311TAAGGAGACTAGGTTCCAATAGCTGGGCTCATCGATGACCC N39/PCA3 AAGATGGCGGC non-promN40b 312 TTACACAAATCGTGGGTTGGCCTCTGGCTCATCGATGACCC N40/PCA3 AAGATGGCGGCnon-prom N41b 313 CGAAAGCGTTCCGCAGGACCCCCTTGGCTCATCGATGACCC N41/PCA3AAGATGGCGGC non-prom N42b 314 AAAGTCTGAGAATGAGTGATACCATGGCTCATCGATGACCCN42/PCA3 AAGATGGCGGC non-prom N43b 315ATATTGGTAGTTTTGTCCGCTGTAGGGCTCATCGATGACCC N43/PCA3 AAGATGGCGGC non-promN44b 316 CGGAAGATCTAATCTGCACGCAATTGGCTCATCGATGACCC N44/PCA3 AAGATGGCGGCnon-prom N45b 317 GCGCCTCGTTGGGCAGAAGTTTGTGGAAATGGCTCATCGAT N45/PCA3GACCCAAGATGGCGGC non-prom N46b 318ATCTTCACCTACCGAGTTCTACGGGCCTACGGCTCATCGAT N45/PCA3 GACCCAAGATGGCGGCnon-prom N47b 319 CCCACAACTTGCACCCGCTATGCGACCCTGGGCTCATCGAT N47/PCA3GACCCAAGATGGCGGC non-prom N48b 320GATTAGTGGCCCAACGGGAACAAACTTCCTGGCTCATCGAT N48/PCA3 GACCCAAGATGGCGGCnon-prom N49b 321 CGCCCGTCCCAGACCCTTACTCACTATGGAGGCTCATCGAT N49/PCA3GACCCAAGATGGCGGC non-prom N50b 322GAGATTGTACCCTACAGTCCGATTACCGATGGCTCATCGAT N50/PCA3 GACCCAAGATGGCGGCnon-prom N51b 323 CGCAGTAAAAGGGCACAGGTAATTACCTTAGGCTCATCGAT N51/PCA3GACCCAAGATGGCGGC non-prom N52b 324AGGGTGTCTTGAACTACTGGCGCAGCCCATGGCTCATCGAT N52/PCA3 GACCCAAGATGGCGGCnon-prom N53b 325 TCGGCGGCGGGTAGTCAGTTCGCTACCTGGGGCTCATCGAT N53/PCA3GACCCAAGATGGCGGC non-prom N54b 326GATGTGATCTGGACCCTACGGGAGGGGACAGGCTCATCGAT N54/PCA3 GACCCAAGATGGCGGCnon-prom N55b 327 AGTCCCAGATATGAGAGAAGCGAAGCATAAGGCTCATCGAT N55/PCA3GACCCAAGATGGCGGC non-prom N56b 328ACTATTACACCACGTACCGTAGGTCGGCTCATCGATGACCC N56/PCA3 AAGATGGCGGC non-prom329 GTCATATGCGACGATCTCAGGGCTCATCGATGACCCAAGAT U20/PCA3 GGCGGC 330TCTCCCTATAGTGAGTCGTATTAAATTGTCATATGCGACGA RC TCTCAG PROM/U20 PCA3 U20-331 TCTCCCTATAGTGAGTCGTATTAAATTGTCATATGCGACGA RC cPROTCTCAGGGCTCATCGATGACCCAAGATGGCGGC Prom/U20/ PCA3 (NOTE: PCA3 sequencesame as non- promoter primer) 332GTGATGCTTTATGAGATTCCGGTCTCCGACGGCTCATCGAT N5/PCA3 GACCCAAGATGGCGGC 333TCTCCCTATAGTGAGTCGTATTAAATTGTGATGCTTTATGA RC PROM/N5 GATTCCGGTCTCCGACN5b_cPRO 334 TCTCCCTATAGTGAGTCGTATTAAATTGTGATGCTTTATGA RCGATTCCGGTCTCCGACGGCTCATCGATGACCCAAGATGGCG Prom/N5/ GC PCA3 335TTCATCGAGGTACATTGGTGCTATTCCATTGGCTCATCGAT N10/PCA3 GACCCAAGATGGCGGC 336TCTCCCTATAGTGAGTCGTATTAAATTTTCATCGAGGTACA RC TTGGTGCTATTCCATT PROM/PCA3N10_cPRO 337 TCTCCCTATAGTGAGTCGTATTAAATTTTCATCGAGGTACA RCTTGGTGCTATTCCATTGGCTCATCGATGACCCAAGATGGCG Prom/N10/ GC PCA3 338GATGTCCTAAAATGAGGCGTGGCAATAGAGGGCTCATCGAT N12/PCA3 GACCCAAGATGGCGGC 339TCTCCCTATAGTGAGTCGTATTAAATTGATGTCCTAAAATG RC AGGCGTGGCAATAGAG PROM/N12N12_cPRO 340 TCTCCCTATAGTGAGTCGTATTAAATTGATGTCCTAAAATG RCAGGCGTGGCAATAGAGGGCTCATCGATGACCCAAGATGGCG Prom/N12/ GC PCA3 341CAGAGTCATGTATACCCACTGTCGGTCGAAGGCTCATCGAT N13/PROM GACCCAAGATGGCGGC 342TCTCCCTATAGTGAGTCGTATTAAATTCAGAGTCATGTATA RC CCCACTGTCGGTCGAA PROM/N13N13b_cPRO 343 TCTCCCTATAGTGAGTCGTATTAAATTCAGAGTCATGTATA Prom/N13/CCCACTGTCGGTCGAAGGCTCATCGATGACCCAAGATGGCG PCA3 GC 344ATGTTATGGAGAGTGGGTTAGGCAAGGCTCATCGATGACCC N28/PCA3 AAGATGGCGGC 345TCTCCCTATAGTGAGTCGTATTAAATTATGTTATGGAGAGT RC PROM GGGTTAGGCAA PCA3N28b_cPRO 346 TCTCCCTATAGTGAGTCGTATTAAATTATGTTATGGAGAGT RCGGGTTAGGCAAGGCTCATCGATGACCCAAGATGGCGGC Prom/N28/ PCA3 347AAAGTCTGAGAATGAGTGATACCATGGCTCATCGATGACCC N42/PCA3 AAGATGGCGGC 348TCTCCCTATAGTGAGTCGTATTAAATTAAAGTCTGAGAATG RC AGTGATACCAT RPOM/N42N42b_cPRO 349 TCTCCCTATAGTGAGTCGTATTAAATTAAAGTCTGAGAATG RCAGTGATACCATGGCTCATCGATGACCCAAGATGGCGGC Prom/N42/ PCA3 350CCCACAACTTGCACCCGCTATGCGACCCTGGGCTCATCGAT N47/PCA3 GACCCAAGATGGCGGC 351TCTCCCTATAGTGAGTCGTATTAAATTCCCACAACTTGCAC RC CCGCTATGCGACCCTG PROM/N47N47b_cPRO 352 TCTCCCTATAGTGAGTCGTATTAAATTCCCACAACTTGCAC RCCCGCTATGCGACCCTGGGCTCATCGATGACCCAAGATGGCG  Prom/N47/ GC PCA3 353GATTAGTGGCCCAACGGGAACAAACTTCCTGGCTCATCGAT N48/PCA3 GACCCAAGATGGCGGC 354TCTCCCTATAGTGAGTCGTATTAAATTGATTAGTGGCCCAA RC CGGGAACAAACTTCCT PROM/N48N48b_cPRO 355 TCTCCCTATAGTGAGTCGTATTAAATTGATTAGTGGCCCAA RCCGGGAACAAACTTCCTGGCTCATCGATGACCCAAGATGGCG Prom/N48/ GC PCA3 356CGCCCGTCCCAGACCCTTACTCACTATGGAGGCTCATCGAT N49/PCA3 GACCCAAGATGGCGGC 357TCTCCCTATAGTGAGTCGTATTAAATTCGCCCGTCCCAGAC RC CCTTACTCACTATGGA PROM/N49N49b_cPRO 358 TCTCCCTATAGTGAGTCGTATTAAATTCGCCCGTCCCAGAC RCCCTTACTCACTATGGAGGCTCATCGATGACCCAAGATGGCG Prom/N49/ GC PCA3 BOmePCA3 359GAUGCAGUGGGCAGCUGUGAGGAC PCA3 3e3(−) 112- BLOCKER 3′blk RPCA3e3(−) 360UGUGUCUUCAGGAUGAAACACACA PCA3  147-166_C9 PROBE (21/22) 361AUCUGUUUUCCUGCCCAUCCUUUAAG PCA3 TARGET CAPTURE PCA3e4(−) 362AUCUGUUUUCCUGCCCAUCCUUUAAGTTTAAAAAAAAAAAA PCA3 109dT3A30 AAAAAAAAAAAATARGET 3′-blocked CAPTURE 363 CCACTGCATCAGGAACAAAAGCGTGATCTTG PSA target specific sequence for promoter primer 364TGGCTAATCCCGCCACTGCATCAGGAACAAAAGCGTGATCT T1/PSA TG T1 365GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCG Prom/T1 PSA T1b 366GGCTCATCGATGACCCAAGATGGCGGCTGGCTAATCCCGCC Prom/T1/ACTGCATCAGGAACAAAAGCGTGATCTTG PSA 367AGGTTGAGTCCGCATCTGAACCACTGCATCAGGAACAAAAG T18/PSA CGTGATCTTGG T18 368GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18 TCTGAA PSA T18b 369GGCTCATCGATGACCCAAGATGGCGGCAGGTTGAGTCCGCA Prom/T18/TCTGAACCACTGCATCAGGAACAAAAGCGTGATCTTG PSA 370ATGTGCGCACAACCACTGCATCAGGAACAAAAGCGTGATCT T7/PSA TG T7 371GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAA Prom/T7 PSA T7b 372GGCTCATCGATGACCCAAGATGGCGGCATGTGCGCACAACC Prom/T7/ACTGCATCAGGAACAAAAGCGTGATCTTG PSA 373TATGAATGCGACCCGGAACCACTGCATCAGGAACAAAAGCG T15/PSA TGATCTTG T15 374GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15 GGAA PSA T15b 375GGCTCATCGATGACCCAAGATGGCGGCTATGAATGCGACCC Prom/T15/GGAACCACTGCATCAGGAACAAAAGCGTGATCTTG PSA 376CATGTACCAACGCCACTGCATCAGGAACAAAAGCGTGATCT T3/PSA TG T3 377GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACG Prom/T3 PSA T3b 378GGCTCATCGATGACCCAAGATGGCGGCCATGTACCAACGCC Prom/T3/ACTGCATCAGGAACAAAAGCGTGATCTTG PSA 379AATCTCCGAGCGCCACTGCATCAGGAACAAAAGCGTGATCT T9/PSA TG T9 380GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCG Prom/T9 PSA T9b 381GGCTCATCGATGACCCAAGATGGCGGCAATCTCCGAGCGCC Prom/T9/ACTGCATCAGGAACAAAAGCGTGATCTTG PSA 382TCACCATGTACCAACGCCACTGCATCAGGAACAAAAGCGTG T14/PSA ATCTTG T14 383GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14 CG PSA T14b 384GGCTCATCGATGACCCAAGATGGCGGCTCACCATGTACCAA Prom/T14/CGCCACTGCATCAGGAACAAAAGCGTGATCTTG PSA 385AACAATGGTCACTGCATCCCACTGCATCAGGAACAAAAGCG T16/PSA TGATCTTG T16 386GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16 CATC PSA T16b 387GGCTCATCGATGACCCAAGATGGCGGCAACAATGGTCACTG Prom/T16/CATCCCACTGCATCAGGAACAAAAGCGTGATCTTG PSA 388GGGCCGTTTCCGGACATAACCACTGCATCAGGAACAAAAG T17/PSA CGTGATCTTG T17 389GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17 ACATAA PSA T17b 390GGCTCATCGATGACCCAAGATGGCGGCGGGCCGTTTCCCGG Prom/T17/ACATAACCACTGCATCAGGAACAAAAGCGTATCTTG PSA 391TGAAAGAGTTGTCAGTTTGCTGGTCCACTGCATCAGGAACA T21/PSA AAAGCGTGATCTTG T21 392GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21 GTTTGCTGGT PSA T21b393 GGCTCATCGATGACCCAAGATGGCGGCTGAAAGAGTTGTCA Prom/T21/GTTTGCTGGTCCACTGCATCAGGAACAAAAGCGTGATCTTG PSA 394TCAGGTAAAGGTTCCTCACGCTACCCCACTGCATCAGGAAC T22/PSA AAAAGCGTGATCTTG T22395 GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22 CTCACGCTACCPSA T22b 396 GGCTCATCGATGACCCAAGATGGCGGCTCAGGTAAAGGTTC Prom/T22/CTCACGCTACCCCACTGCATCAGGAACAAAAGCGTGATCTT PSA G 397GCTGTGGCTGACCTGAAATACC PSA target specific sequence for non- promoterprimer PSA N5b 398 GTGATGCTTTATGAGATTCCGGTCTCCGACGCTGTGGCTGA N5/PSACCTGAAATACC non-prom PSA N12b 399GATGTCCTAAAATGAGGCGTGGCAATAGAGGCTGTGGCTGA N12/PSA CCTGAAATACC non-promPSA N13b 400 CAGAGTCATGTATACCCACTGTCGGTCGAAGCTGTGGCTGA N13/PSACCTGAAATACC non-prom PSA N28b 401ATGTTATGGAGAGTGGGTTAGGCAAGCTGTGGCTGACCTGA N28/PSA AATACC non-promPSA N42b 402 AAAGTCTGAGAATGAGTGATACATGCTGTGGCTGACCTGA N42/PSA AATACCnon-prom PSA N47b 403 CCCACAACTTGCACCCGCTATGCGACCCTGGCTGTGGCTGA N47/PSACCTGAAATACC non-prom PSA N48b 404GATTAGTGGCCCAACGGGAACAAACTTCCTGCTGTGGCTGA N48/PSA CCTGAAATACC non-promPSA N49b 405 CGCCCGTCCCAGACCCTTACTCACTATGGAGCTGTGGCTGA N49/PSACCTGAAATACC non-prom RPSAe2e3(−) 406 GAUGCAGUGGGCAGCUGUGAGGAC PSA 222-BLOCKER 244_BKD RPSAe3(−) 407 UGUGUCUUCAGGAUGAAACACACA PSA PROBE32-51_C9 (19, 20) 408 CGAACUUGCGCACACACGUCAUUGGA PSA  TARGET CAPTUREPSA(−) 409 CGAACUUGCGCACACACGUCAUUGGATTTAAAAAAAAAAAA PSA  581dT3A30AAAAAAAAAAAAAAAAAA TARGET CAPTURE Keys to identity of sequences:Normal-tag sequences Bold-pca3 target specific sequencesDouble Underline-PSA target specific sequences Underline-promotersequence Italics-dT(3)A(30) tail

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

The invention claimed is:
 1. A method for identifying a nucleic acid tag sequence for use in a nucleic acid assay, comprising: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences; b) screening the pool of nucleic acid sequences to identify two or more nucleic acid sequences having two or more performance characteristics; c) selecting one or more nucleic acid sequences, each for use as tag sequence in a nucleic acid assay; db) comparing a nucleic acid sequence or sequences from the pool of nucleic acid sequences against a database having one or more nucleic acid sequences to determine complementarity of the nucleic acid sequences from the pool of nucleic acid sequences to the database having one or more sequences, ec) generating a sub-pool of nucleic acid sequences, wherein the sub-pool is a collection of nucleic acid sequences with complementarity that is less than 95% to the nucleic acid sequence(s) in the database, that is less than 90% to the nucleic acid sequence(s) in the database; that is less than 80% to the nucleic acid sequence(s) in the database, that is less than 70% to the nucleic acid sequence(s) in the database, or that is less than 50% to the nucleic acid sequence(s) in the database; fd) screening the sub-pool of nucleic acid sequences for one or more performance characteristics selected from melting temperature, activity in an enzyme reaction, G-C content, nucleobase composition, length, hybridization energy, multimer formation, internal structure formation, G-quartet formation, and hairpin-stability, ge) selecting one or moreat least one nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay; hf) synthesizing at least two different oligonucleotides for use in a nucleic acid assay, wherein each of the synthesized oligonucleotides has a tag sequence selected according to step ge); and ig) measuring for each of the different oligonucleotides synthesized in step hf) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay, and optionally comparing the measurements to the measurements obtained for an untagged oligonucleotide without the tag sequence, wherein the tags of the different oligonucleotides do not serve as binding sites for amplification oligomers in the nucleotide acid assay; and jh) selecting one or more of the nucleic acid tag sequences used in step ig)for use in a nucleic acid assay; ki) modifying the sequence of thea tag sequence incorporated into an oligonucleotide from step h) to obtain a modified tag sequence for incorporation into an oligonucleotide; lj) measuring for the oligonucleotide containing a modified tag sequence from step ki) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and mk) selecting one or more of the modified nucleic acid tag sequences used in step ij) for use in a nucleic acid assay.
 2. The method according to claim 1, wherein the modification in step k) comprises systematically deleting nucleotides from the tag sequence.
 3. The method according to claim 1, further comprising the steps of: (i) modifying the sequence of the tag sequence from step g); (ii) synthesizing an oligonucleotide to contain the modified tag sequence; (iii) measuring for the oligonucleotide containing a modified tag sequence one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay; and (iv) selecting one or more of the modified nucleic acid tag sequences used in step (iii) for use in a nucleic acid assay.
 4. The method according to claim 3 1, wherein the modification in step (i) comprises systematically deleting nucleotides from the tag sequence.
 5. The method according to claim 1, wherein; the performance characteristic(s) comprises one or more performance characteristic(s) selected from the group consisting of: amplification performance characteristic(s); interference with nucleic acids in the nucleic acid assay; interference with one or more oligonucleotides in the nucleic acid assay; interference with one or more target nucleic acids in the nucleic acid assay; interference with one or more amplicons in the nucleic acid assay; assay reproducibility; quantification; real-time quantification; end-point quantification; a dynamic range for detecting target nucleic acid; a limit of detection; precision of replicates; reaction kinetics; and a combination thereof.
 6. The method according to claim 1, wherein: a nucleic acid sequence in the pool sub-pool is used as a tag in a the nucleic acid assay of step g) and reduces interference with a nucleic acid in the nucleic acid assay to about 95% or less compared to the amount of interference present in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in an in vitro nucleic acid assay and accelerates reaction kinetics to about 105% or more compared to the reaction kinetics in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in an in vitro nucleic acid assay and slows reaction kinetics to about 95% or less compared to the reaction kinetics in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and increases sensitivity for a target nucleic acid so that the amount of target nucleic acid needed to obtain a detectable signal is about 95% or less of the amount of target nucleic acid required in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and decreases sensitivity for a target nucleic acid so that the amount of target nucleic acid needed to obtain a detectable signal is about 105% or more of the amount of target nucleic acid required in an untagged assay; or a nucleic acid sequence in the pool is used as a tag in a nucleic acid assay and increases replication precision by about 105% or more compared to an untagged assay.
 7. The method according to claim 6 1, wherein the nucleic acid assay of step g) is an in vitro isothermal amplification assay, or the nucleic acid assay is an in vitro PCR amplification assay, or the nucleic acid assay is a sequencing assay, which generates an amplification product of the assay incorporating one of the tag sequences, which is used to identify the amplification product.
 8. The method according to claim 6, wherein the tag is part of an amplification oligomer.
 9. The method according to claim 6, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein, or wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.
 10. The method according to claim 1, wherein the tag sequence has sequences selected in step e) have a Tm that is less than or equal to 72° C.; or wherein the tag sequence has sequences have a Tm selected from the group consisting of all whole and partial numbers from 35° C. through 75° C.; or wherein the tag sequence has sequences have a primer dimer energy formation that is less than or equal to −10.0 kcal/mol; or wherein the tag sequence has sequences have a hairpin stability energy that is less than or equal to −4 kcal/mol; or wherein the 3′ region of the tag sequence sequences is less than 80% complementary to the one or more oligonucleotides in the searched database; or a combination thereof.
 11. The method according to claim 1, wherein the nucleic acid assay comprises two or more target nucleic acids; or wherein the nucleic acid assay comprises a target nucleic acid combined from two or more separate samples; or wherein the nucleic acid assay comprises a target nucleic acid combined from two or more separate samples and the target nucleic acid from each separate sample includes a unique tag sequence to identify from which samples the target nucleic acid originated.
 12. The method according to claim 1, wherein the database having one or more nucleic acid sequences is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof; or wherein the database having one or more nucleic acid sequences is a database containing sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof.
 13. A method for identifying nucleic acid tag sequences for use in an in vitro nucleic acid amplification assay, comprising the steps of: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences from Table 1; b) screening the pool of nucleic acid sequences against a database containing one or more nucleic acid sequences to identify percent complementarity between nucleic acid sequences in the pool and nucleic acid sequences in the database; c) screening the pool of nucleic acid sequences to determine a performance characteristic selected from the group consisting of: G-C content, nucleobase composition, length, multimer formation, primer-dimer formation, Tm, hairpin stabilization energy, self dimer stabilization energy, internal structure formation, G-quartet formation, hybridization energy, activity in an enzyme reaction, and combinations thereof d) generating a sub-pool of nucleic acid sequences from the results obtained in step b), step c) or steps b) and c); e) selecting one or more nucleic acid sequences from the sub-pool for use as tag sequences in a nucleic acid assay; f) synthesizing an amplification oligomer containing a tag sequence selected at step e); and g) performing an in vitro nucleic acid amplification reaction using the amplification oligomer.
 14. The method according to claim 13, wherein the sub-pool at step d) contains one or more of the following: nucleic acid sequences with Tm values that are within ±2 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±5 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with Tm values that are within ±10 degrees C. from a mean Tm of nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±5% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±10% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents that are within ±30% from the mean G-C content of the nucleic acids in the sub-pool; nucleic acid sequences with G-C contents from 30% to 80%, from 40% to 70%, or from 30% to 50%; nucleic acid sequences in Table 2; and nucleic acid sequences with lengths from 5 nucleobases to 100 nucleobases.
 15. The method according to claim 13, wherein the in vitro amplification reaction performed at step g) has one or more of the following performance characteristics: reduced interference between nucleic acids in the reaction when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; reaction kinetics that are accelerated by about 105% or more when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; reaction kinetics that are reduced to about 95% or less when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer; increased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 95% or less than the minimum amount of starting material required in an untagged assay in order to obtain a detectable signal; decreased sensitivity when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer, wherein the in vitro amplification reaction using the tagged amplification oligomer requires an amount of starting material that is about 105% or more than the amount of starting material required in an untagged assay in order to obtain a detectable signal; and a replication precision that is about 105% or better when performed with the tagged amplification oligomer from step f) compared to when performed using an untagged amplification oligomer.
 16. The method according to claim 15, wherein the tagged assay decreases the performance parameter by from 25% to 94%, from 50% to 94%, or from 75% to 94% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein; or wherein the tagged assay increases the performance parameter by from 105% to 150%, from 105% to 200%, or from 105% to 500% compared to the untagged assay, wherein each range is inclusive of all whole and partial numbers therein.
 17. The method according to claim 13, wherein the one or more nucleic acid sequences in a database is a collection of various nucleic acid sequences corresponding to a nucleic acid assay, a public collection of nucleic acid sequences, an aligned collection of nucleic acid sequences, the pool of nucleic acid sequences, or a combination thereof; or wherein the one or more nucleic acid sequences in a database contains sequence(s) that are derived from: collections of various nucleic acid sequences corresponding to a nucleic acid assay; a public collection of nucleic acid sequences; a collection of aligned sequences, the pool, or a combination thereof.
 18. The method according to claim 13, wherein the in vitro amplification assay is an isothermal amplification assay; or a multiplex amplification assay; or a PCR amplification reaction; or a combination thereof.
 19. The method according to claim 18, wherein an amplicon generated in the in vitro amplification assay is used in a sequencing assay.
 20. A tagged amplification oligomer containing a tag sequence obtained by the method of claim
 13. 21. A multiplex in vitro amplification reaction mixture containing one or more tagged amplification oligomers, each with a tag sequence obtained by any the method of claim
 13. 22. A kit for amplification of a target nucleic acid, wherein the kit contains at least one tagged amplification oligomer containing a tag sequence obtained by the method of claim
 13. 23. A collection of nucleic acid sequences useful as tag sequences for use in a nucleic acid assay, wherein the collection contains at least two sequences in Table 1 or the collection is Table 1, Table 2 or Table
 3. 24. A method for identifying a nucleic acid tag sequence for use in a nucleic acid assay, comprising: a) generating a pool of nucleic acid sequences, wherein the pool is at least three nucleic acid sequences; b) screening the pool of nucleic acid sequences to identify two or more nucleic acid sequences, each having two or more performance characteristics; and c) selecting one or more nucleic acid sequences with the two or more performance characteristics identified in step b), each for use as tag sequence in a nucleic acid assay; wherein step b) comprises: i) comparing a nucleic acid sequence or sequences from the pool of nucleic acid sequences against a database having one or more nucleic acid sequences to determine complementarity of the nucleic acid sequences from the pool of nucleic acid sequences to the database having one or more sequences; ii) generating a sub-pool of the nucleic acid sequences of step i), wherein the sub-pool is a collection of nucleic acid sequences with complementarity that is less than 95% to the nucleic acid sequence(s) in the database; iii) screening the sub-pool of nucleic acid sequences for one or more performance characteristics selected from melting temperature, activity in an enzyme reaction, G-C content, nucleobase composition, length, hybridization energy, multimer formation, internal structure formation, G-quartet formation, and hairpin-stability; iv) synthesizing at least two different oligonucleotides for use in a nucleic acid assay, wherein each of the synthesized oligonucleotides has a tag sequence selected according to step iii); and v) measuring for each of the different oligonucleotides synthesized in step iv) one or more of the following performance characteristics: speed of amplification, limit of detection, interference, precision of replicates, performance against a specific target nucleic acid sequence, or performance against multiple target nucleic acid sequences in a nucleic acid assay, and comparing the measurements to measurements obtained for an oligonucleotide lacking the tag, wherein the tags of the different oligonucleotides do not serve as binding sites for amplification oligomers in the nucleic acid assay, thereby identifying one or more nucleic acid tags sequences for use in a nucleic acid assay.
 25. The method of claim 24, wherein step v) measures an improved performance characteristic for at least one of the different oligonucleotides relative to a corresponding untagged oligonucleotide.
 26. The method of claim 24, wherein the nucleic acid assay of step v) comprises performing an in vitro isothermal amplification assay, an in vitro PCR amplification assay, or a sequencing assay with an amplification oligomer comprising a nucleic acid tag sequence identified in step v), which generates an amplification product incorporating one of the modified tag sequences, which is used to identify the amplification product. 